Neural and behavioral control in Caenorhabditis elegans by a yellow-light–activatable caged compound

Caenorhabditis elegans is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist N-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving C. elegans without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

Caged compounds that release bioactive molecules upon light irradiation have been widely used for photocontrol of cell signaling (1). Various molecules such as neurotransmitters, nucleotides, ions, drugs, fluorophores, and proteins can be rendered biologically inert by using photoreactive caging groups (2). Light irradiation induces photolysis of the caging groups to restore the bioactivity of these molecules. Since the initial applications of caged cyclic adenosine monophosphate (cAMP) (3) and caged adenosine triphosphate (ATP) (4) in biological experiments, caged compounds have been applied to cultured cells (1), brain slices (5, 6), and living animals (7). Although the light-mediated delivery of chemical probes in vivo is challenging, there are reports of photoactivation of neurons in Drosophila by caged ATP (8, 9) and photo-mediated gene activation in zebrafish by caged RNA/DNA (10, 11). The optical transparency of these species makes them particularly suitable targets for photochemical probes.The nematode Caenorhabditis elegans is also amenable to optical manipulation using photocontrollable tools owing to its transparent body, compact nervous system, and ease of genetic manipulation (12–15). Nevertheless, few reports describe the application of caged compounds to C. elegans. One reason for this may be that most conventional caged compounds have the major limitation that uncaging requires short-wavelength (ultraviolet to blue) light, which induces an innate photophobic response, as well as causing cell damage or even death of the nematode (16, 17). Thus, it would be preferable to achieve photocontrol by using longer-wavelength visible light.Methods for uncaging with longer-wavelength visible light (green to near infrared) include photorelease via metal–ligand photocleavage (18), via photooxidative C-C cleavage and hydrolysis (19), and by using a photosensitizer (20). However, these strategies have disadvantages: The photocages can only release the fluorophore upon irradiation at over 550 nm (18), the caged compounds show relatively poor temporal resolution of neural control due to the multistep nature of the photoreaction (19), and they generate toxic levels of reactive oxygen species under normoxic conditions (20). Therefore, they are unsuitable for neurophysiological experiments in C. elegans, a model system that is widely used to elucidate the neuronal basis of behavior. Another method for releasing bioactive molecules with visible/near-infrared light is to use two-photon excitation, wherein the excitation wavelength can be twice that of the one-photon counterpart (21–23). Two-photon excitation is particularly useful for studies that require spatially high-resolution uncaging, such as functional mapping of receptors along dendrites (6) and single-spine stimulation (24), although the amount of the photorelease is very small (25).Here, we aimed to develop practically useful caged compounds that can be uncaged by one-photon excitation in C. elegans without the disadvantages described above and that would be suitable for neurophysiological experiments. We confirmed that our compounds exhibit sharp absorption spectra at around 580 nm that do not overlap with those of GFP-based probes and show simple, single-step photorelease of the caged molecules in response to light irradiation at over 550 nm. We also validated the application of caged N-vanillylnonanamide for neurophysiological studies. Uncaging by illumination at 580 nm with simultaneous monitoring of neuronal activity using GCaMP demonstrated that the uncaging triggers responses in sensory neurons, body wall muscles, motor neurons, and interneurons that are associated with behavioral changes of freely moving C. elegans.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

A neural circuit for competing approach and defense underlying prey capture

Predators must frequently balance competing approach and defensive behaviors elicited by a moving and potentially dangerous prey. Several brain circuits supporting predation have recently been localized. However, the mechanisms by which these circuits balance the conflict between approach and defense responses remain unknown. Laboratory mice initially show alternating approach and defense responses toward cockroaches, a natural prey, but with repeated exposure become avid hunters. Here, we used in vivo neural activity recording and cell-type specific manipulations in hunting male mice to identify neurons in the lateral hypothalamus and periaqueductal gray that encode and control predatory approach and defense behaviors. We found a subset of GABAergic neurons in lateral hypothalamus that specifically encoded hunting behaviors and whose stimulation triggered predation but not feeding. This population projects to the periaqueductal gray, and stimulation of these projections promoted predation. Neurons in periaqueductal gray encoded both approach and defensive behaviors but only initially when the mouse showed high levels of fear of the prey. Our findings allow us to propose that GABAergic neurons in lateral hypothalamus facilitate predation in part by suppressing defensive responses to prey encoded in the periaqueductal gray. Our results reveal a neural circuit mechanism for controlling the balance between conflicting approach and defensive behaviors elicited by the same stimulus.

The ability to seek and capture prey adeptly is a conserved behavior essential to the survival of numerous animal species. However, attacking prey brings risks for the predator, and efficient hunting requires a skillful balance between responding to threatening and appetitive prey cues. The neural circuits involved in this balance are unknown. Rodents naturally hunt and consume a variety of insects, and hunting behavior has been studied in the laboratory using rats given cockroaches or mice (1) and, more recently, in laboratory mice given crickets (2–5). In early studies, immediate early gene mapping during hunting identified the recruitment of lateral hypothalamus (LHA) (1, 6, 7) and periaqueductal gray (PAG) (7, 8), and electrical stimulation of either LHA (9–12) or PAG (13, 14) could initiate avid predatory attacks in rats and cats, suggesting the presence of neuronal cell populations that promote hunting in both these brain structures.LHA has been historically regarded as a central node in the neural system controlling seeking behaviors, including feeding and predatory hunting (14–16). Recent circuit neuroscience work has confirmed this role by finding that stimulation of Vgat+ GABAergic neurons in LHA can trigger predation as well as feeding (5, 17–20). However, the role of these neurons in predation may be indirect, as any manipulation that increases food seeking is expected to promote motivation to hunt as well. Moreover, some studies in which LHA GABAergic neurons were optogenetically stimulated did not observe the full repertoire of feeding behaviors but instead found increased chewing activity, digging, or general motor activity (21–23), suggesting that the link between LHA-driven feeding and hunting may be more complex. One possible confound in these studies is their use of two different Cre driver lines (Vgat::Cre versus Gad2::Cre) that have been shown to target distinct LHA GABAergic neurons (23). Thus, it remains unclear to what extent different populations of LHA GABAergic neurons specifically encode and control predatory behaviors.A link between LHA and PAG in predation was made by a recent study showing that activation of LHA to PAG projections can promote predatory hunting in mice (5), and the central importance of the PAG in hunting is further strengthened by several studies identifying a series of PAG afferents arising from different brain structures—including the central nucleus of the amygdala, medial preoptic area, and zona incerta—that promote predation (3, 4, 24, 25). However, the precise functional role of the PAG in hunting remains unclear. For example, it was found that inhibition of glutamatergic neurons in the lateral and ventrolateral PAG (l/vlPAG) by GABAergic inputs from the central nucleus of the amygdala promotes predatory behavior (3), suggesting that PAG neurons might function to suppress rather than promote predation. At the same time, other studies have shown that activation of glutamatergic neurons in l/vlPAG induces defensive behaviors (26), a finding that is consistent with an extensive literature linking PAG to both innate and learned defensive behavior (27–29). One explanation for these observations is that PAG has an antagonistic role in hunting by promoting defensive behaviors toward prey early in the encounter when familiarity with the prey is low. Suppression of these defensive responses by GABAergic inputs would therefore facilitate unimpeded predation.Here, we identify Gad2+ neurons in LHA as those specifically recruited during predatory chasing and attack, and both are sufficient and necessary to drive hunting, but not feeding or social behavior, in male mice. Activation of LHA Gad2+ neuron projections to PAG decreased defensive responses to prey and promoted hunting during the early phases of predation when male mice learned to hunt cockroaches. Finally, in vivo calcium endoscopy identified a neural population in PAG that encoded risk assessment and flight behaviors elicited by prey. Notably, we failed to detect PAG neurons responsive to predatory pursuit or attack. These results point to a circuit in which activity in LHA Gad2+ neurons promotes hunting in part by suppressing defensive responses encoded in PAG.     

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

To ensure survival under unfavorable environmental conditions and synchronize populations, most insect species enter diapause, which is a seasonal adaptation that evolved as a specific subtype of dormancy (1, 2). Diapause is not a passive response to changing conditions but rather an actively induced state that precedes adverse natural situations. Therefore, this diapause phenotype is accompanied by changes in energy metabolism or storage to improve cold/stress tolerance in later life stages, or progeny via reproductive switch (3). Although it has been generally suggested that brain/neuroendocrine systems are associated with this seasonal reproductive plasticity in both vertebrates and invertebrates (3, 4), the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration into morphological, physiological, behavioral, and reproductive responses, known as the diapause syndrome, remains unclear (3).The silkworm Bombyx mori is a typical insect that arrests normal development during early embryogenesis, which is accompanied by metabolic changes in diapause (5, 6). The development of diapause-destined embryos is arrested during the G2 cell cycle stage immediately after the formation of the cephalic lobe and telson and sequential segmentation of the mesoderm (7). The bivoltine strain of B. mori has two generations per year, and progeny diapause is transgenerationally induced as a maternal effect and is determined by the environmental temperature, photoperiod, and nutrient conditions during embryonic and larval development of the mother (5, 6). The temperature signal during the mother’s embryonic development predominantly affects diapause determination, even if silkworms of the bivoltine Kosetsu strain are exposed to all cases of photoperiods during embryonic and larval development. In the Kosetsu strain, when eggs are incubated at 25 °C under continuous darkness, the resultant female moths (25DD) lay diapause eggs in almost all cases. In contrast, incubation of eggs at 15 °C in dark condition results in moths (15DD) that lay nondiapause eggs in almost all cases (6).Embryonic diapause is induced by the diapause hormone (DH) signaling pathway, which consists of highly sensitive and specific interactions between a neuropeptide, DH, and DH receptor (DHR) (6, 8). DH is exclusively synthesized in seven pairs of neurosecretory cells (DH-PBAN–producing neurosecretory cells [DHPCs]) located within the subesophageal ganglion (SG) in the mother’s generation (6). DH is released into the hemolymph during pupal–adult development and acts on the DHR, which belongs to the G protein-coupled receptors (GPCRs) (9). DH levels in the hemolymph are higher in the 25DD than 15DD pupae in the middle of pupal–adult development when the developing ovaries are sensitive to DH (6). Furthermore, the embryonic Bombyx TRPA1 ortholog (BmTRPA1) acts as a thermosensitive channel that is activated at temperatures above ∼21 °C and affects diapause induction through DH release (10). However, there remain questions about the thermal information that is received by BmTRPA1 and linked to DH signaling to induce diapause.From the 1950s, it has been suggested that the DH release was controlled by signals derived from certain region(s) in the brain based on surgical experiments, such as midsagittal bisection or transection (11–13). Especially, the operation in nondiapause producers changed them to diapause producers while transection of the protocerebrum had no effect on the diapause producers. These surgical results suggested the involvement of the protocerebrum in the inhibitory control of DH secretion (12, 14). Furthermore, the accumulation of the ovarian 3-hydroxykynurenine (3-OHK) pigment that accompanies the diapause syndrome was affected by injection with γ-aminobutyric acid (GABA) and the plant alkaloid picrotoxin (PTX), which is a widely used ionotropic GABA and glycine receptor antagonist (15, 16), and the selective ionotropic GABA receptor (GABAR) antagonist bicuculline. This suggests that a GABAergic neurotransmission via ionotropic GABAR is involved in DH secretion, which may be active in nondiapause producers but inactive in diapause producers throughout the pupal–adult development (14, 17). In general, ionotropic GABAR is composed of homo- or hetero-pentameric subunits. All known GABAR subunits display a similar structural scheme, with a large N-terminal extracellular domain involved in the formation of a ligand-binding pocket and a pore domain made of four transmembrane alpha-helices (TM1–TM4) (16, 18). Four homologous sequences of the ionotropic GABAR subunit genes were identified as RDL, LCCH3, GRD, and a GRD-like sequence named 8916 in various insects (19). However, the in vivo physiological roles of both signals derived from the brain and the GABAergic pathway in diapause induction have not been previously investigated.Corazonin (Crz) is an undecapeptide neurohormone sharing a highly conserved amino acid (a.a.) sequence across insect lineages and is involved in different physiological functions, such as heart contraction (20), stress response (21, 22), various metabolic activities (23–25), female fecundity (26), melanization of locust cuticles (27), regulation of ecdysis (28, 29), and control of caste identity (30). Moreover, Crz belongs to the gonadotropin-releasing hormone (GnRH) superfamily alongside adipokinetic hormone (AKH) and AKH/Crz-related peptide (ACP). Duplicates of an ancestral GnRH/Crz signaling system occurred in a common ancestor of protostomes and deuterostomes through coevolution of the ligand receptor (31, 32).Herein, we demonstrated that the hierarchical pathway consists of a GABAergic and Crz signaling system modulating progeny diapause induction by acting on DH release. We propose that the PTX-sensitive GABAergic signal may act to chronically suppress Crz release in dorsolateral Crz neurons (under nondiapause conditions) and that diapause conditions (or PTX injection) inhibits GABAergic signaling, resulting in accelerated Crz release, which in turn induces DH release. GABA signaling may be finely tuned by the temperature-dependent expression of the plasma membrane GABA transporter (GAT), which differs between the 25DD and 15DD conditions. Furthermore, this signaling pathway possesses similar features to the GnRH signaling system with respect to seasonal reproductive plasticity in vertebrates.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

A microbial metabolite synergizes with endogenous serotonin to trigger C. elegans reproductive behavior

Natural products are a major source of small-molecule therapeutics, including those that target the nervous system. We have used a simple serotonin-dependent behavior of the roundworm Caenorhabditis elegans, egg laying, to perform a behavior-based screen for natural products that affect serotonin signaling. Our screen yielded agonists of G protein-coupled serotonin receptors, protein kinase C agonists, and a microbial metabolite not previously known to interact with serotonin signaling pathways: the disulfide-bridged 2,5-diketopiperazine gliotoxin. Effects of gliotoxin on egg-laying behavior required the G protein-coupled serotonin receptors SER-1 and SER-7, and the G_q ortholog EGL-30. Furthermore, mutants lacking serotonergic neurons and mutants that cannot synthesize serotonin were profoundly resistant to gliotoxin. Exogenous serotonin restored their sensitivity to gliotoxin, indicating that this compound synergizes with endogenous serotonin to elicit behavior. These data show that a microbial metabolite with no structural similarity to known serotonergic agonists potentiates an endogenous serotonin signal to affect behavior. Based on this study, we suggest that microbial metabolites are a rich source of functionally novel neuroactive molecules.

In the vertebrate brain, the neurotransmitter serotonin is produced by a small number of neurons that project widely to regulate diverse neural circuits. Accordingly, a large number of psychiatric diseases are treated with small-molecule therapeutics that agonize or antagonize serotonin signaling (1, 2). Small molecules that boost endogenous serotonin signaling, such as inhibitors of serotonin catabolism or reuptake and serotonin receptor agonists, are used to treat major depression, anxiety disorders, and alcohol and nicotine addiction (3–5). Compounds that inhibit serotonin receptors are also clinically important; many antipsychotics are potent antagonists of a subset of G protein-coupled serotonin receptors. Although the links between serotonin and psychiatric disease have been firmly established and several classes of small-molecule therapeutics that target serotonin signaling systems are available in the brain, there is still a need for new small-molecule agonists and antagonists of serotonin signaling. Many patients do not respond to available therapeutics, which can also cause undesired side effects (6, 7).The tiny roundworm Caenorhabditis elegans offers the opportunity to use high-throughput behavior-based screens to discover small molecules that target serotonin signaling. The C. elegans nervous system uses serotonin to generate simple and stereotyped behaviors. One such behavior is egg laying. A pair of serotonergic neurons—the hermaphrodite specific motor neurons (HSNs)—innervate egg-laying muscles (ELMs) (8). HSNs are necessary for normal egg laying, and activation of HSNs is sufficient to trigger egg-laying behavior. This behavior is particularly well suited for high-throughput screening because it leaves a visible trace that obviates the need to observe the behavior in real time: eggs that have been released into the environment. Also, the egg-laying circuit is readily accessible to pharmacological agents. Exogenous serotonin stimulates egg laying (9, 10) as do many canonical regulators of serotonin signaling that are in clinical use: e.g., serotonin-reuptake inhibitors and receptor agonists (10–13). Importantly, the C. elegans egg-laying system uses molecular mechanisms of serotonin signaling that have counterparts in the vertebrate brain. HSNs use conserved pathways for the synthesis, storage, and release of serotonin (14–18). G protein-coupled serotonin receptors that are homologous to mammalian HTR2 and HTR7 mediate activation of ELMs (19, 20), and these nematode serotonin receptors signal via conserved G_q and G_s subunits to activate highly conserved second-messenger signaling cascades. Mutants exist for many of the key components of the serotonin signaling pathway that promote egg laying. This permits genetic analysis of mechanisms of drug action, which can accelerate the process of matching novel compounds to their biological targets.Here, we report the identification of a microbial metabolite, the 2,5-diketopiperazine (DKP) gliotoxin, as a potent activator of C. elegans egg-laying behavior. Genetic studies of gliotoxin sensitivity and physiological measurements of the effects of gliotoxin on serotonin neurons and their targets indicate that gliotoxin potentiates signaling via G_q-coupled serotonin receptors in a manner that strictly depends on the presence of serotonin. Although this is reminiscent of serotonin-reuptake inhibitors such as imipramine and fluoxetine, which work by amplifying endogenous serotonin signals, gliotoxin does not require serotonin-reuptake transporters for its effects on behavior, indicating that it acts via a novel mechanism. Our data suggest that gliotoxin-like compounds might constitute a useful class of neuroactive small molecules with therapeutic potential. We further suggest that microbial metabolites are an unexplored resource for neuroactive small molecules with new and useful properties, and we discuss the possibility that these molecules arise during coevolution of microbes and soil-dwelling nematodes such as C. elegans.     

Discriminating symbiosis and immunity signals by receptor competition in rice

Plants encounter various microbes in nature and must respond appropriately to symbiotic or pathogenic ones. In rice, the receptor-like kinase OsCERK1 is involved in recognizing both symbiotic and immune signals. However, how these opposing signals are discerned via OsCERK1 remains unknown. Here, we found that receptor competition enables the discrimination of symbiosis and immunity signals in rice. On the one hand, the symbiotic receptor OsMYR1 and its short-length chitooligosaccharide ligand inhibit complex formation between OsCERK1 and OsCEBiP and suppress OsCERK1 phosphorylating the downstream substrate OsGEF1, which reduces the sensitivity of rice to microbe-associated molecular patterns. Indeed, OsMYR1 overexpression lines are more susceptible to the fungal pathogen Magnaporthe oryzae, whereas Osmyr1 mutants show higher resistance. On the other hand, OsCEBiP can bind OsCERK1 and thus block OsMYR1–OsCERK1 heteromer formation. Consistently, the Oscebip mutant displayed a higher rate of mycorrhizal colonization at early stages of infection. Our results indicate that OsMYR1 and OsCEBiP receptors compete for OsCERK1 to determine the outcome of symbiosis and immunity signals.

In nature, plants live and interact with diverse microbes, including symbionts and pathogens. To discern friends from foes, plants have evolved various receptors that sense external microbes. Plant immune responses are triggered when pattern recognition receptors (PRRs) at the plasma membrane recognize microbe-associated molecular patterns (MAMPs) (1). MAMPs are highly conserved molecular signatures within a class of microbes and include fungal chitin, bacterial flagellin, and elongation factor Tu (EF-Tu) (2). PRRs comprise receptor-like kinases and receptor-like proteins (3). In plant–symbiont interactions, receptor kinases at the plasma membrane recognize signals that trigger symbiosis (4, 5).Arbuscular mycorrhizal (AM) fungi secrete short-chain chitooligosaccharides (COs) and nonsulfated lipochitooligosaccharides (LCOs), called mycorrhizal factors (Myc factors), that are recognized by plant receptors and mediate the establishment of AM symbiosis (6–14). In rice, perception of the AM symbiotic signal is mediated by a lysin motif (LysM)–containing receptor kinase (LYKs), OsMYR1, that directly binds to CO4 and subsequently interacts with OsCERK1 (8). Interestingly, OsCERK1 is also a well-known receptor involved in MAMP-triggered immunity (15–17). In rice, an OsCERK1–OsCEBiP receptor complex recognizes chitin and triggers immune responses (18). Additionally, OsCERK1 interacts with OsLYP4 and OsLYP6 to participate in peptidoglycan perception (19). Thus, OsCERK1 is a node that crosses immunity and symbiosis.Fungal cell walls consist of about 1 to 20% chitin, which is a long-chain polymer of N-acetylglucosamine. To protect themselves from fungal infection, plants secrete chitinases that break down chitin and release COs (20). Long-chain COs are recognized by specific receptors and trigger immunity, whereas short-chain COs are associated with non-stress–related plant responses (21). Similarly, in mammals, shorter oligomers induce a weaker defense response than longer oligomers (22). Intriguingly, although chitin is the principal component of AM fungi, AM symbiosis triggers only a weak defense response (23, 24). Moreover, pretreatment of plants with CO4 also suppresses their defense response (21), implying that CO4 and OsMYR1 might suppress defense responses during AM symbiosis. However, the mechanism remains unclear.Interestingly, a recent study reported that COs ranging from four to eight residues in length (CO4 to CO8) can serve as symbiotic signals in Medicago truncatula, although CO8 is typically considered an immunity signal (9, 25). In rice, LCOs cannot induce symbiotic calcium oscillations, and short-chain COs are the major symbiotic signals from mycorrhizal fungi (26). In this study, we found that the shorter-chain chitooligosaccharide CO4 and its receptor OsMYR1 can suppress immune signaling induced by CO8 in rice. Our data indicate that the balanced perception of CO4 and CO8 by the symbiotic receptor OsMYR1, and the MAMP receptor OsCEBiP is crucial for the establishment of AM symbiosis in rice.     

A complement factor H homolog,heparan sulfation,and syndecan maintain inversin compartment boundaries in C. elegans cilia

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the developed world, affecting 11% of adults over the age of 85. Although the discovery of a Y402H variant in complement factor H (CFH) as a major risk factor for AMD marked a major advance for the field, the roles of CFH and Y402H in the mechanisms of disease initiation and progression remain unclear (1–5).The alternative pathway is one of three complement system pathways that are part of the innate immune system''s natural defense against infections. The canonical function of CFH in modulating alternative complement pathway activity suggests a central role for excessive alternative complement pathway activity in the progressive photoreceptor dysfunction that is characteristic of AMD pathogenesis (1–8). CFH is a secreted protein composed of 20 complement control protein (CCP) repeats (also known as short complement repeats (SCRs) or sushi domains) that inhibits alternative complement pathway-associated inflammation and cytolysis on host cell surfaces by promoting decay of the convertase that cleaves and activates C3 and also by acting as a cofactor for factor I in processing C3b to its inactive form (9).CFH interacts with host cell surfaces by binding to heparan sulfate (HS) through binding sites found in CCP 7 and CCP 19 to 20 (10). HSs are heterogenous repeats of glucuronic acid and N-acetylglucosamine disaccharide covalently attached to proteoglycan core proteins that may be transmembrane such as the syndecans, glycosylphosphatidylinositol-anchored proteins such as the glypicans, or secreted such as perlecan, agrin, and collagen XVIII (11–14).HS heterogeneity results from variation in disaccharide length and partial deacetylation, epimerization, and modification by 2-, 3-, and 6-O sulfotransferases (15). Sequence variants within HS binding sites in CFH, including Y402H in CCP 7, result in reduced HS affinity, suggesting that increased AMD risk is a result of reduced CFH on cell surfaces and increased alternative complement pathway activity within the retina (16).However, CFH is also reported to have functions outside its canonical role in regulating the alternative complement pathway that include preventing lipid binding to HS and modulating monocyte migration (17, 18). This suggests that noncanonical mechanisms may contribute to the dysfunction and death of photoreceptor cells that are associated with AMD progression. In addition to increased AMD-risk, the CFH Y402H variant is also associated with increased risk for a defect in rod-mediated dark adaptation (RMDA) that is detectable prior to other clinical AMD symptoms. This suggests that a neuronal signaling defect involving CFH may be an early contributor to AMD-associated neuronal dysfunction (19). In support of this concept, many of the clinical symptoms associated with AMD can be reproduced by defects in structural and signaling proteins associated with photoreceptor outer segments, which are modified primary cilia (20, 21).Primary cilia are highly compartmentalized microtubule-based protrusions with conserved compositions and organizations containing (from proximal to distal) a transition zone, middle, and distal segment [TZ, MS, DS (22)]. Within the MS, a number of cilia subtypes in mammals and invertebrates (including Caenorhabditis elegans) possess a domain called the “inversin compartment,” defined by a specific localization of the ciliopathy protein, inversin [also called NPHP2 (23–25)]. Defects in inversin/NPHP-2 may cause left-right body asymmetry for which inversin is named and the early onset cystic kidney disease, nephronophthisis-2 [NPHP-2 (23, 26)]. Within the inversin compartment, inversin/NPHP-2 assembles into filaments of variable length depending on cell type, suggesting the presence of an unknown mechanism, not bound by an obvious structure or membrane, that regulates inversin/NPHP-2 filament length (27, 28).Here, we find that a C. elegans structural homolog of CFH localizes on a subset of mechanosensory neuron primary cilia in a syndecan- and heparan 3-O sulfotransferase–dependent manner where it prevents inversin/NPHP-2 from accumulating in distal cilia compartments in aging adults. Similar observations that CFH restricts ectopic inversin/NPHP-2 accumulation in vertebrate photoreceptors suggests that previously unappreciated defects in cilia organization may contribute to photoreceptor dysfunction in AMD patients with CFH mutations in HS binding sites.     

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae. Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Multidrug-resistant (MDR) bacterial infections have become a major threat to human health (1–3). Mortality rates from infections caused by gram-negative bacteria, specifically Klebsiella pneumoniae, are on the rise owing to the lack of effective antibiotics to treat the emergent MDR strains (4–7). The capsule of K. pneumoniae is composed of extracellular polysaccharides that promote infection by masking the bacteria from immune recognition and provide an especially potent barrier against peptide-based antimicrobials, including innate host defense peptides and last-resort polymyxin antibiotics (8–14).Antimicrobial peptides are commonly amphipathic, with both a charged and a hydrophobic character (15). The anionic nature of the bacterial capsule promotes an electrostatic attraction to cationic antimicrobial peptides, and peptide hydrophobicity has been proposed to enhance capsule binding through nonionic interactions (9, 12, 16). Interaction with the bacterial capsule is thought to induce structural changes that cause sequestration of antimicrobial peptides to prevent them from reaching their bacterial membrane target (16, 17). While the bacterial capsule inhibits host defense peptides and polymyxins, a few amphipathic antimicrobial peptides have been identified that can retain activity against capsulated K. pneumoniae (18–21). However, it is not known what enables some peptides to avoid sequestration by the capsule of K. pneumoniae while the capsule effectively neutralizes our innate host defense peptides with similar physicochemical properties. This lack of knowledge prevents us from understanding how to bypass the capsule barrier that K. pneumoniae uses to avoid our innate immune response and last-resort treatment options.Here we characterize the synthetic evolution of a peptide inhibited by capsule to a peptide with potent activity against capsulated K. pneumoniae. Remarkably, our results indicate that rather than reduced interactions, our active peptide retains binding to capsule and undergoes conformational changes associated with capsule aggregation. We present a model in which peptide-driven sequestration of capsule disrupts this barrier and reduces its ability to protect K. pneumoniae against antimicrobial attack. These findings provide insight into improving antimicrobial peptide activity against K. pneumoniae and may help strengthen our understanding of the inability of innate host defense peptides to act on capsulated bacteria.     

Molecular switch architecture determines response properties of signaling pathways

Many intracellular signaling pathways are composed of molecular switches, proteins that transition between two states—on and off. Typically, signaling is initiated when an external stimulus activates its cognate receptor that, in turn, causes downstream switches to transition from off to on using one of the following mechanisms: activation, in which the transition rate from the off state to the on state increases; derepression, in which the transition rate from the on state to the off state decreases; and concerted, in which activation and derepression operate simultaneously. We use mathematical modeling to compare these signaling mechanisms in terms of their dose–response curves, response times, and abilities to process upstream fluctuations. Our analysis elucidates several operating principles for molecular switches. First, activation increases the sensitivity of the pathway, whereas derepression decreases sensitivity. Second, activation generates response times that decrease with signal strength, whereas derepression causes response times to increase with signal strength. These opposing features allow the concerted mechanism to not only show dose–response alignment, but also to decouple the response time from stimulus strength. However, these potentially beneficial properties come at the expense of increased susceptibility to upstream fluctuations. We demonstrate that these operating principles also hold when the models are extended to include additional features, such as receptor removal, kinetic proofreading, and cascades of switches. In total, we show how the architecture of molecular switches govern their response properties. We also discuss the biological implications of our findings.

Several molecules involved in intracellular signaling pathways act as molecular switches. These are proteins that can be temporarily modified to transition between two conformations, one corresponding to an on (active) state and another to an off (inactive) state. Two prominent examples of such switches are proteins that are modified by phosphorylation and dephosphorylation and GTPases that bind nucleotides. For phosphorylation–dephosphorylation cycles, it is common for the covalent addition of a phosphate by a kinase to cause activation of the modified protein. A phosphatase removes the phosphate to turn the protein off. In the GTPase cycle, the protein is on when bound to guanosine triphosphate (GTP) and off when bound to guanosine diphosphate (GDP). The transition from the GDP-bound state to the GTP-bound state requires nucleotide exchange, whereas the transition from the GTP-bound to the GDP-bound state is achieved via hydrolysis of the γ phosphate on GTP. The basal rates of nucleotide exchange and hydrolysis are often small. These reaction rates are increased severalfold by Guanine Exchange Factors (GEFs) and GTPase Accelerating Proteins (GAPs), respectively (1, 2).A signaling pathway is often initiated upon recognition of a stimulus by its cognate receptor, which then activates a downstream switch. In principle, a switch may be turned on by three mechanisms: (a) activation, by increasing the transition rate from the off state to the on state; (b) derepression, by decreasing the transition rate from the on state to the off state; and (c) concerted activation and derepression. Examples of these three mechanisms are found in the GTPase cycles in different organisms. In animals, signaling through many pathways is initiated by G-protein-coupled receptors (GPCRs) that respond to a diverse set of external stimuli. These receptors act as GEFs to activate heterotrimeric G proteins (3–6). Thus, pathway activation relies upon increasing the transition rate from the off state to the on state. There are no GPCRs in plants and other bikonts; the nucleotide exchange occurs spontaneously, without requiring GEF activity (7–9). G proteins are kept in the off state by a repressor such as a GAP or some other protein that holds the self-activating G protein in its inactive state. In this scenario, the presence of a stimulus results in derepression, i.e., removal of the repressing activity (10–12). Concerted activation and dererpression occur in the GTPase cycle of the yeast mating-response pathway (13, 14), in which the inactive GPCRs recruit a GAP protein and act to repress, whereas active receptors have GEF activity and act to activate. Thus, perception of a stimulus leads to concerted activation and derepression by increasing GEF activity while decreasing GAP activity.These three mechanisms of signaling through molecular switches also occur in many other systems. For example, the activation mechanism described here is a simpler abstraction of a linear signaling cascade, a classical framework used to study general properties of signaling pathways (15–19), as well as to model specific signaling pathways (20–22). While derepression may seem like an unusual mechanism, it occurs in numerous important signaling pathways in plants (e.g., auxin, ethylene, gibberellin, and phytochrome), as well as gene regulation (23–27). In many of these cases, derepression occurs through a decrease in the degradation rate of a component instead of its deactivation rate. Concerted mechanisms are found in bacterial two-component systems, wherein the same component acts as kinase and phosphatase (28–35).Many previous studies have focused on the properties of a single switch mechanism without drawing comparisons between the three potential ways for initiating signaling. For example, the classical Goldbeter–Koshland model studied zero-order ultrasensitivity of an activation mechanism (15). Further analyses examined the effect of receptor numbers (36–38), feedback mechanisms (39, 40), and removal of active receptors via endocytosis and degradation (41, 42). Similarly, important properties of the concerted mechanism have been elucidated, such as its ability to perform ratiometric signaling (13, 14), to align dose responses at different stages of the signaling pathway (43), as well as its robustness (29, 44). The derepression mechanism is relatively less studied. Although there are models of G-signaling in Arabidopsis thaliana (45–47), these models have a large number of states and parameters and do not specifically examine distinct behaviors conferred by derepression.What are the evolutionary constraints that may favor activation over derepression and vice versa? Seminal studies have investigated this question for gene-regulatory networks (48–50). However, an analysis of differences in the functional characteristics of activation, derepression, and concerted mechanisms in the context of cell signaling is still lacking. To address this deficiency, we perform a systematic comparison of the three mechanisms using the following metrics: 1) dose–response, 2) response time, and 3) ability to suppress or filter stochastic fluctuations in upstream components. The rationale behind comparing dose–response curves is that they provide information about the input sensitivity range and the output dynamic range, both of which are of pharmacological importance. We supplement this comparison with response times, which provide information about the dynamics of the signaling activity. The third metric of comparison is motivated from the fact that signaling pathways are subject to intrinsic fluctuations that occur due to the stochastic nature of biochemical reactions (51–56).We construct and analyze both deterministic ordinary differential equation (ODE) models and stochastic models based on continuous-time Markov chains. We show that activation has the following two effects: It makes the switch response more sensitive than that of the receptor, and it speeds up the response with the stimulus strength. In contrast, derepression makes the switch response less sensitive than the receptor occupancy and slows down the response speed as stimulus strength increases. These counteracting behaviors of activation and derepression lead to intermediate sensitivity and intermediate response time for the concerted mechanism. In the special case of a perfect concerted mechanism (equal activation and repression), the dose–response curve of the pathway aligns with the receptor occupancy, and the response time does not depend upon the stimulus level. The noise comparison reveals that the concerted mechanism is more susceptible to fluctuations than the activation and derepression mechanisms, which perform similarly. We further show that these results qualitatively hold for more complex models, such as those incorporating receptor removal and proofreading. We finally discuss our findings to suggest reasons that might have led biological systems to evolve one of these mechanisms over the others, a question that has received considerable attention in the context of gene regulation (48–50).     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

Spatial distribution of LTi-like cells in intestinal mucosa regulates type 3 innate immunity

Lymphoid tissue inducer (LTi)-like cells are tissue resident innate lymphocytes that rapidly secrete cytokines that promote gut epithelial integrity and protect against extracellular bacterial infections.Here, we report that the retention of LTi-like cells in conventional solitary intestinal lymphoid tissue (SILT) is essential for controlling LTi-like cell function and is maintained by expression of the chemokine receptor CXCR5. Deletion of Cxcr5 functionally unleashed LTi-like cells in a cell intrinsic manner, leading to uncontrolled IL-17 and IL-22 production. The elevated production of IL-22 in Cxcr5-deficient mice improved gut barrier integrity and protected mice during infection with the opportunistic pathogen Clostridium difficile. Interestingly, Cxcr5^−/− mice developed LTi-like cell aggregates that were displaced from their typical niche at the intestinal crypt, and LTi-like cell hyperresponsiveness was associated with the local formation of this unconventional SILT. Thus, LTi-like cell positioning within mucosa controls their activity via niche-specific signals that temper cytokine production during homeostasis.

Lymphoid tissue inducer (LTi)-like cells belong to a family of tissue resident innate lymphocytes that lack rearranged antigen-specific receptors and act as a first line of defense at barrier tissues. LTi-like cells, along with other group 3 innate lymphoid cells (ILC3), maintain intestinal homeostasis by producing the cytokines IL-22 and IL-17A, which promote gut epithelial cell proliferation, anti-microbial peptide production, and tight junction protein abundance (1, 2). The conditioning of epithelial cells by these cytokines contributes to balanced interactions between the host and commensal microbiota under steady-state conditions, and LTi-like cell-derived IL-22 promotes barrier integrity and protective immunity during infection with the enteric pathogenic bacteria (3).In addition to providing effector functions, LTi-like cells and their fetal LTi counterparts are required for early steps in lymphoid tissue development. Fetal LTi induce lymph node and Peyer’s patch development during gestation by activating lymphoid tissue organizer cells at primordial lymphoid organs with lymphotoxin (LT)-α1β2 (4–6). Similarly, LTi-like cells are required for the postnatal development of cryptopatches, small lymphoid aggregates in the intestine that have the potential to mature into isolated lymphoid follicles (ILF) in response to signals from microbes (7, 8). In line with their roles in lymphoid tissue organogenesis and maturation, LTi-like cells in adult mouse intestines preferentially localize in solitary intestinal lymphoid tissue (SILT). The microenvironments of these highly specialized niches are expected to support and regulate LTi-like cells; however, their impact on LTi-like cell behavior has not been fully explored.LTi-like cells express multiple G protein–coupled receptors that facilitate their migration in tissue (9–12). Among these, CXCR5 has a predominant role in the migration of LTi to developing lymphoid structures, with Cxcr5^−/− mice exhibiting defects in lymph node and Peyer’s patch development (13). Mice deficient in CXCR5 or its ligand CXCL13 also have delayed cryptopatch development and fail to convert cryptopatches to mature ILF because of impaired recruitment of B cells to these structures (14–16). Dendritic cells (DCs) have been shown to be a local source of CXCL13 in SILT (16) and thus likely retain B cells and LTi-like cells at these structures under homeostatic conditions via the CXCL13–CXCR5 signaling axis. The retention of LTi-like cells in SILT is expected to bring these cells in close proximity to activating and inhibitory signals provided by specialized myeloid cells, neurons that express the vasoactive intestinal peptide (VIP), and lymphocyte populations localized at these sites (17–20). However, the impact of CXCR5 on functions of LTi-like cells beyond those associated with lymphoid tissue maintenance and development remains unknown.In the current study, we show that CXCR5 expression regulates LTi-like cell function. Deletion of Cxcr5 led to increased numbers of LTi-like cells in the small intestine (SI) and enhanced their ability to produce IL-17A and IL-22. Cxcr5 regulated LTi-like cells via a cell-intrinsic mechanism that did not involve direct suppression by CXCL13. Heightened LTi-like cell activity in Cxcr5-deficient mice was associated with the development of abnormal LTi-like cell aggregates in the SI that were localized in villus lamina propria instead of at the intestinal crypt base. Importantly, augmented production of IL-22 in Cxcr5^−/− mice was protective during acute infection with the opportunistic pathogen Clostridium difficile. These data reveal that CXCR5-dependent migration can control innate type 3 immunity by altering the niche of LTi-like cells in intestinal lamina propria.     

Immune cells fold and damage fungal hyphae

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans–macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin–myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.

An estimated 1.5 million people succumb to a systemic fungal infection each year (1). Most of these individuals had HIV or had undergone a medical intervention that severely compromised their immunity. The innate immune system plays a key role in preventing fungal infection (2–4). The efficacy of these defenses depends on the outcome of individual interactions between innate immune cells and fungal pathogens.Innate immune cells such as macrophages recognize fungal pathogens via pattern recognition receptors (PRRs) that interact with pathogen-associated molecular patterns (PAMPs), many of which lie at the fungal cell surface (5, 6). The formation of a phagocytic synapse between PRRs and PAMPs triggers the active engulfment of the pathogen and subsequent attempts to kill the fungal cell using a variety of mechanisms that include a toxic mix of reactive chemical species and antimicrobial peptides (2). Meanwhile, fungal pathogens attempt to evade immune recognition, phagocytosis, and killing through a range of strategies that include PAMP masking to reduce recognition (7, 8), robust stress responses to attenuate the potency of reactive oxygen and nitrogen species (9–11), the activation of pyroptosis to kill the immune cells (12–14), and in particular, cellular morphogenesis (15–18). Candida albicans activates morphogenetic programs to form hyphae that are challenging to phagocytose and clear, not least because of their extreme length (6, 15, 16). Hypha formation also provides a means of fungal escape from the macrophage (12, 13).While examining dynamic interactions between macrophages and fungal cells, we observed that these immune cells can fold fungal hyphae. We reasoned that this fungal folding must involve the application of mechanical forces and that this folding contributes to fungal clearance. Therefore, we examined the involvement of the cytoskeletal network and PRRs in this phenomenon, providing initial clues as to how macrophages anchor a fungal hypha and achieve leverage to fold it.     

Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the Survival of Motoneuron 1 gene (SMN1) and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a Caenorhabditis elegans SMA model showed less expression of the B-Raf homolog lin-45. Accordingly, motoneuron survival was rescued by a cell autonomous lin-45 expression in a C. elegans SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of newborns, infants, and young adults which preferentially affects lower motoneurons in the ventral horn of the spinal cord. As a consequence, patients suffer from muscle weakness and atrophy often resulting in respiratory insufficiency and early death. SMA is caused by homozygous deletions or mutations of the Survival of Motoneuron 1 (SMN1) gene (1). However, humans harbor the similar SMN2 gene which codes for the same protein but differs in a critical cytosine to thymine exchange within exon seven in an exonic splice enhancer region (2). Consequently, the SMN2 pre-mRNA is insufficiently spliced, resulting in low levels of functional full-length mRNA and protein (3). Thus, SMA is caused by low SMN protein levels. The number of SMN2 gene copies critically modifies the disease phenotype with a low number associated with severe forms and higher numbers with milder forms (4).SMA is characterized by a neuromuscular phenotype starting in proximal muscles with hypotonia, fatigue, and paralysis. The disease is categorized in five different subtypes based on the clinical presentation (5). A small number of patients suffer from congenital SMA-Type 0 with a prenatal onset and a rapid disease progression. The majority of the patients are classified as severe Type 1. The onset is postnatal between 2 wk and 6 mo of age. Untreated patients are never able to roll or sit independently, and about two-thirds decease within the first 2 y of life. Type 2 patients develop first symptoms between 6 and 18 mo of age and are unable to walk independently. Type 3 patients have mild progressive muscle weaknesses with a normal life-expectancy, and Type 4 patients have some difficulties with gross motor functions only (5).However, recently approved treatments dramatically changed the clinical situation. Nusinersen, an antisense oligonucleotide, and Risdiplam, a small molecule compound, both correct SMN2 pre-mRNA splicing (6–8). Onasemnogene Abeparvovec is a gene-therapy based on the delivery of a SMN cDNA by an adeno-associated virus (AAV) (9, 10). All treatments substantially enhance the survival and motor functions which will enhance the SMA prevalence. However, clinical and preclinical studies demonstrate that delayed interventions after disease onset led to limited clinical improvements. Moreover, there are a substantial number of nonresponders (7, 11, 12). Onasemnogene Abeparvovec, Nusinersen, and Risdiplam enhance the SMN-protein level thereby termed SMN-dependent treatments. Since SMN levels have already been restored by those drugs, other complementary therapies are needed which do not change the SMN levels: SMN-independent approaches (13). An intervention in the pathomechanisms downstream of SMN reduction is a promising strategy for the development of such SMN-independent approaches. Therefore, it is crucial to understand those mechanisms.We and others previously identified dysregulated cellular signaling as potential mechanisms of motoneuron degeneration in SMA. Such pathways include the Rho kinase (ROCK) (14–16), the extracellular-regulated kinase (ERK) (17–19), the tumor protein p53 (20, 21), p38 mitogen-activated protein kinase (22), and the c-Jun N-terminal kinase 3 (JNK3) pathways (23), all of which were up-regulated in SMA. However, it is not clear whether and how those pathways interfere with each other. We previously showed a bidirectional crosstalk of the ERK and the ROCK pathways being important for the pathophysiology of SMA mice (18, 19). An isolated and reductionist view on single pathways may indeed obscure a global view on altered signaling in SMA. Network biology allows such a global view, which provides a basis for an informed decision for the most important master regulator of dysregulated signaling and motoneuron degeneration in SMA (13).Here, we present a network biology approach for the identification of such critical signaling nodes within a network of altered signaling in a SMA mouse model. We identifed B-Raf and 14-3-3 ζ/δ as elements of a central and highly connected signaling hub in an SMA network. It has been previously shown that this is an important signaling hub which provides motoneuron function and survival in response to neurotrophic factors. B-Raf was reduced in SMA mice spinal cords already at presymptomatic stages. This reduction localizes to murine SMA motoneurons and was confirmed in SMA patient-derived motoneurons. Accordingly, we could rescue motoneuron loss and locomotion deficiency in vivo in a Caenorhabditis elegans SMA model by re-expression of the B-Raf homolog lin-45. Inhibition of MEK and ERK homologs, downstream targets of B-Raf, abrogated the neuroprotective function of B-Raf in C. elegans. Importantly, the rescue was obtained after onset of neurodegeneration and in a cell-autonomous, motoneuron-intrinsic manner.     

Diverse innate stimuli activate basophils through pathways involving Syk and IκB kinases

Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE cross-linking but also by endogenous “innate” factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFβ, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases.

Basophils are rare circulating granulocytes activated by immunoglobulin E (IgE)-mediated cross-linking of the high affinity receptor for IgE (FcεRI), which induces their degranulation and synthesis of the type 2 cytokines, interleukin 4 (IL-4) and IL-13 in autoimmune, allergic diseases and helminthiases (1). Basophils are also sensitive to innate signals, including the cytokine IL-3, the alarmins IL-18, IL-33, and Thymic stromal Lymphopoietin (TSLP), the prostaglandin D2 (PGD2), ATP, and various chemokines or growth factors (1–4). Some of these stimuli might regulate basophil immunoregulatory functions during homeostasis (5). Secretion of IL-4 and histamine release by human basophils is sensitive to FK506 (a calcineurin inhibitor), while secretion of IL-13 is not affected, indicating that distinct signaling pathways regulate the expression of these type 2 cytokines (6–8). By contrast, activation of basophils by IgE or IL-18/33 involves distinct receptors and upstream signaling pathways but results in expression of both IL-4 and IL-13 (5, 7, 9–11). Despite common evolutionary ancestry, it is currently thought that IL-4 and IL-13 show divergent functions in immunity and physiology (12, 13). Basophil IL-4 secretion has been implicated in T helper type 2 (Th2) differentiation and M2 skewing. Basophils are an important source of IL-13 in the lungs, where they control the phenotype of alveolar macrophages during development (1, 5).The Il4/13 locus is subject to a differential regulation between distinct cell types but has been mostly studied in T cells. The locus contains elements regulated by cAMP, c-fos/AP-1, NFAT, NF-κB, GATA3, STAT5, and STAT6 that have been associated with the regulation of Il4/13 expression (6, 14–19). Basophils produce IL-4 and IL-13 after the cross-linking of their surface-bound IgE by antigen or by exposure to IL-3. IgE- or IL-3-mediated basophil activation are both controlled by the tyrosine kinase Syk (6, 20, 21). IgE-induced cytokine expression is also promoted by IκB kinase (IKK) through NF-κB-dependent or -independent signals (22, 23).To date, studies of murine basophils have focused on cultured bone marrow-derived basophils (BMBa) or primary bone marrow (BM) basophils, with circulating mature basophils too few in number for useful studies (6). We used B8 × 4C13R IL-4/IL-13 triple reporter mice (24) to follow and compare the ex vivo responses of primary mature basophils to 96 common stimuli and found that basophils were highly sensitive to type 2 and epithelial-derived cytokines and growth factors. Single-cell analysis revealed that basophils displayed distinct phenotypes upon stimulation with IL-3, expressing IL-4, and/or IL-13, Ly6C, or the degranulation marker CD63 (25). During helminth infection, basophils were found to be hyper- and hyporeactive to distinct stimuli (“Hp-basophils”). Purified Hp-basophils were also sensitive to homeostatic growth factors and antiviral response elements. Basophil reactivity was always sensitive to Syk and IKKs specific inhibitors. In conclusion, we found basophils to be sensitive to a complex array of distinct innate stimuli that worked through core common signaling pathways.