From the Cover: The point of no return in vetoing self-initiated movements

In humans, spontaneous movements are often preceded by early brain signals. One such signal is the readiness potential (RP) that gradually arises within the last second preceding a movement. An important question is whether people are able to cancel movements after the elicitation of such RPs, and if so until which point in time. Here, subjects played a game where they tried to press a button to earn points in a challenge with a brain–computer interface (BCI) that had been trained to detect their RPs in real time and to emit stop signals. Our data suggest that subjects can still veto a movement even after the onset of the RP. Cancellation of movements was possible if stop signals occurred earlier than 200 ms before movement onset, thus constituting a point of no return.It has been repeatedly shown that spontaneous movements are preceded by early brain signals (1–8). As early as a second before a simple voluntary movement, a so-called readiness potential (RP) is observed over motor-related brain regions (1–3, 5). The RP was found to precede the self-reported time of the “‘decision’ to act” (ref. 3, p. 623). Similar preparatory signals have been observed using invasive electrophysiology (8, 9) and functional MRI (7, 10), and have been demonstrated also for choices between multiple-response options (6, 7, 10), for abstract decisions (10), for perceptual choices (11), and for value-based decisions (12). To date, the exact nature and causal role of such early signals in decision making is debated (12–20).One important question is whether a person can still exert a veto by inhibiting the movement after onset of the RP (13, 18, 21, 22). One possibility is that the onset of the RP triggers a causal chain of events that unfolds in time and cannot be cancelled. The onset of the RP in this case would be akin to tipping the first stone in a row of dominoes. If there is no chance of intervening, the dominoes will gradually fall one-by-one until the last one is reached. This has been coined a ballistic stage of processing (23, 24). A different possibility is that participants can still terminate the process, akin to taking out a domino at some later stage in the chain and thus preventing the process from completing. Here, we directly tested this in a real-time experiment that required subjects to terminate their decision to move once a RP had been detected by a brain–computer interface (BCI) (25–31).     

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0₄₆₀). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0₄₆₀ was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0₄₆₀ was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0₄₆₀ in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0₄₆₀ in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0₄₆₀ was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.Inherited retinal degenerative diseases affect 1 in 3,000 humans worldwide (1). Retinitis pigmentosa (RP) describes a family of over 50 different gene mutations that cause progressive loss of rod photoreceptors (1, 2). Rod loss is followed by degeneration of cone photoreceptors, ultimately leading to complete blindness in many patients (3). Despite the complete loss of photoreceptors in the outer nuclear layer, many interneurons of the inner retina survive in a functional state for long periods, providing an opportunity for treatment (4, 5).Direct electrical stimulation of the surviving inner retina has proven to be successful in restoring useful vision (6–8). One approach employs surgically implanted photovoltaic or electrode arrays to stimulate retinal ganglion cells (RGCs) (8) or bipolar cells (BCs) (6, 7) directly in the inner nuclear layer (INL) of the degenerated retina, and promising results in clinical trials have led to US Food and Drug Administration approval for the Argus II device (Second Sight Medical Products, Inc.) (8). The electrical implants demonstrate that inner retinal neurons in blind patients can respond to appropriate stimulation and lead to a useful visual percept allowing simple navigation and object recognition. These electronic designs are under continual development to increase the resolution, improve the surgical implantation procedures, and increase the sophistication of their signal-encoding algorithms (9).Microbial opsins, like channelrhodopsin and halorhodopsin, have been successfully tested as visual prosthetics in animal models of human blindness (10–15). Genetically encoded light-gated proteins can be exogenously expressed in retinal cells using viral or nonviral gene delivery vehicles, imparting a light-sensitive function to cone photoreceptors that have become insensitive to light from loss of their outer segments (14), but also to ON-BCs (12, 13), as well as RGCs (10, 15, 16), leading to rescue of basic aspects of visual function in mice. Microbial opsins are appealing for this application due to the bioavailability of the light-sensitive ligand retinal. However, there are potential drawbacks to this approach. Xenotransplantation is generally concerning, because it might lead to immune responses and inflammation potentially spreading to the brain via the optic nerve. Additionally, once expressed, it is impossible to silence the system in case of adverse reactions in patients.One promising alternative to microbial opsins is an optopharmacological strategy that uses synthetic azobenzene-based photoswitches to endow light sensitivity either to native ion channels of neurons (17, 18) or to engineered mammalian receptors and channels that, like the microbial opsins, allow for genetic targeting to specific cells (19–22). We previously showed that an engineered light-gated ionotropic glutamate receptor (LiGluR) restores light responses to blind retina degeneration (rd1) mice (23). The gene encoding for LiGluR was delivered to RGCs by intravitreal injection of adeno-associated virus (AAV), and the photoswitchable tethered ligand maleimide-azobenzene-glutamate (MAG) was delivered in a subsequent intravitreal injection (23). LiGluR contains a single Cys substitution in the kainate receptor, GluK2(439)C, which serves as an anchoring site for MAG close to the ligand binding site. Upon illumination at 380 nm in the near-UV range, the azobenzene linker in MAG photoisomerizes from trans to cis, shortening the molecule and allowing the glutamate to bind into the ligand binding pocket to activate and open the channel.The first-generation MAG photoswitch suffered from two major limitations for vision restoration: (i) The UV light needed for activation is absorbed by the human lens and can damage the retina, and (ii) MAG is bistable, requiring a second pulse of light at a longer wavelength for deactivation. We recently developed a second-generation photoswitch, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0₄₆₀), to overcome these problems (24). MAG0₄₆₀ is activated by white light and spontaneously turns off in the dark. In the present study, we compare retinal light responses and both innate and learned visually guided behaviors in the rd1 mouse model of retina degeneration when LiGluR-MAG0₄₆₀ is targeted to either RGCs or ON-BCs. We find that both cell types support robust light-induced retinal activity and visually guided behavior. To demonstrate efficacy in a larger animal model, we targeted LiGluR-MAG0₄₆₀ to RGCs in a canine model of human blindness and restored light-activated retinal responses in vitro. Because LiGluR-MAG0₄₆₀ is functional in both the mouse and dog, it is an attractive candidate for a genetically encoded retinal prosthetic for the blind.     

Quantitative analysis of vesicle recycling at the calyx of Held synapse

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).Synaptic vesicle recycling ensures synaptic transmission during sustained neuronal activity (1–3). Despite its crucial role, the cycle is poorly understood. In contrast to vesicle exocytosis and endocytosis, which can be directly assayed by presynaptic capacitance measurements and postsynaptic current recordings, vesicle recycling is usually investigated by fluorescence imaging and electron microscopy (EM) with limited signal or temporal resolution (4–7). Likely owing to technical difficulties, the basic properties of vesicle recycling, such as the size of the recycling pool (RP) (3, 6, 8–11), the kinetics of vesicle recycling (6, 8–12), and how the RP supports synaptic transmission (1, 13–15) remain to be elucidated. Classically, presynaptic vesicles can be functionally divided into three populations: the readily releasable pool (RRP), the reserve pool, and the resting pool (3, 16, 17). The RRP is defined as being composed of docked and immediately releasable vesicles (17), which are usually depleted by high-frequency stimulation, prolonged presynaptic depolarization, or the application of hypertonic solution (18–21). The reserve pool functions as a reservoir and serves to maintain vesicle refilling into the RRP (2, 3). These two pools together are commonly referred to as the RP. The resting pool serves as a depot of vesicles for backup use (16, 22). However, it has been debated for a decade whether nerve terminals use the majority (∼100%, from electrophysiology) or only a small fraction (5–40%, from fluorescence imaging and EM) of vesicles in recycling, and whether the RP size undergoes dynamic changes during varied neuronal activity (6, 7, 23–28).The use of vesicles in recycling is a critical determinant of synaptic transmission (1, 13–15). However, it has never been rigorously determined how fast recently recaptured vesicles are organized to recycle and whether vesicles in the RP are homogeneously ready for use (25). Two forms of vesicle retrieval, “kiss-and-run” and full collapse, have been reported for many years. It is still ambiguous whether the rapidly recaptured vesicles in the kiss-and-run mode can be rapidly reused (29–31).Here, we addressed the above issues by developing a new approach to quantify the basic properties of vesicle recycling with unparalleled precision. Different from previous studies in cultured cell systems, the present work combined electrophysiological measurements and EM observations at the calyx of Held synapse in acute brain slices, quantitatively analyzed synaptic vesicle recycling, and kinetically dissected the recycling vesicle pool. We propose a realistic kinetic model and provide new insights into the mechanism that ensures rate-limited but sustainable synaptic transmission.     

Retinal waves regulate afferent terminal targeting in the early visual pathway

Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbian-like processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes long-term impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.The brain employs several strategies to guide the establishment of correct neural connectivity (1, 2). It has been well recognized that the high specificity of connections between the retina and the dorsal lateral geniculate nucleus (dLGN) is established through several factors. These include gradients of axon guidance molecules that guide the initial coarse targeting of afferent terminals (3–6), and spontaneous retinal activity (retinal waves) that drives competitive processes important for the refinement and segregation of afferent terminal branches (2, 7–15).Retinal waves are spontaneous propagating bursts of correlated retinal ganglion cell (RGC) activity and have been classified into three developmental stages (1, 15). Stage II retinal waves (from here on also referred to as retinal waves) are extensively studied and have been found to be critical for the development of retinofugal pathways (1, 2, 15). They are mediated by cholinergic signaling from starburst amacrine cells onto RGCs (8, 13, 16–18) and have been hypothesized to drive the Hebbian-like remodeling of RGC afferent terminals (19, 20). Retinal waves play crucial roles in both the establishment of eye-specific segregation (8, 12, 14, 20, 21), through the removal of afferent branches from opposing putative eye-specific domains, and the refinement of afferent terminals within eye-specific laminae, which is believed to be necessary for the establishment of fine-scale retinotopy (12, 22). However, studies have suggested that retinal waves might play additional roles in the development of the retinogeniculate pathway. When retinal waves are blocked during early development, mature lamination in the adult is abnormal (23–25), while eye-specific segregation recovers (26, 27). These results uncovered a retinal wave-dependent window for the development of retinogeniculate lamination. However, the question remains open as to whether these lamination defects are due to abnormal late eye-specific segregation or the disruption of some form of retinal wave-dependent afferent terminal targeting.A potential retinal wave-dependent mechanism that could regulate retinogeniculate afferent terminal targeting is axon–axon competition originating from the same eye (i.e., intraeye competition). Classic studies in goldfish first demonstrated the principle of axon–axon competition at the optic tectum (28). These studies showed that RGC afferent terminals can undergo expansive or compressive rearrangements in their targeting in response to changes in afferent number, or retinorecipient target size, while maintaining correct retinotopy (28–32). Similarly, neonatal monocular enucleation in ferrets results in an expanded ipsilateral and contralateral projection by adulthood, while correct laminar organization is maintained (7, 10). This demonstrates that retinogeniculate afferent terminals can undergo an expansive and orderly rearrangement due to intraeye competition, and that intereye competition is not required for the establishment of proper retinogeniculate lamination.To investigate whether retinal waves play a role in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, we monocularly enucleated ferrets one day after birth (P1), to eliminate intereye competition, while also pharmacologically blocking retinal waves (P1– P10) in the surviving eye with the cholinergic agonist epibatidine (EPI) (8, 13, 18). Effects on the targeting of retinogeniculate afferents terminals were assessed anatomically, to characterize impacts on retinogeniculate lamination, and functionally, to assess changes in receptive field (RF) structure and retinotopy in the dLGN. Our results demonstrate that retinal waves regulate afferent terminal targeting by way of intraeye competition during the development of the retinogeniculate pathway.     

Neuropsin (OPN5)-mediated photoentrainment of local circadian oscillators in mammalian retina and cornea

The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength–sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.Most mammalian tissues contain autonomous circadian clocks that are synchronized by the suprachiasmatic nuclei (SCN) in the brain (1). The SCN clock itself is entrained by external light cycles through retinal rods, cones, and melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) (2, 3). The retina also manifests a local circadian clock, which regulates many important functions, such as photoreceptor disk shedding, photoreceptor gap-junction coupling, and neurotransmitter release (4–6). Surprisingly, local retinal photoentrainment does not require the SCN, and it also does not require rods, cones, or ipRGCs (7, 8). Thus, the rd1/rd1;Opn4^−/− mouse retina, which has lost essentially all rods and cones due to degeneration and also has an ablated Opn4 gene (3), remains synchronized to light/dark cycles both in vivo and ex vivo (7).To determine the photopigment(s) responsible for local circadian entrainment in the retina, we took a candidate gene approach. Because some cone nuclei may persist in degenerate rd1/rd1 retinas (9), and murine short-wavelength–sensitive cone opsin (OPN1SW) has been reported to be present in the ganglion cell layer (10), we tested the necessity of this pigment for local retinal circadian photoentrainment. We also tested for the involvement of two orphan pigments, encephalopsin (OPN3) (11) and neuropsin (OPN5) (12), both of which are expressed in mammalian retina and, when expressed heterologously, form light-sensitive pigments that activate G proteins (13–17). The function of OPN3 in mammals is unknown despite its widespread expression in neural tissues (18). OPN5 appears to be a deep-brain photopigment in the hypothalamus of birds and is thought to contribute to seasonal reproduction (19–22); it has been immunolocalized to the mammalian inner retina (13, 16) (SI Text); however, to date, no retinal function for this mammalian pigment has been identified. We did not examine two other pigments, retinal G protein-coupled receptor (RGR) opsin (23) and peropsin (RRH) (24). RGR opsin participates in retinoid turnover (25, 26), whereas RRH is expressed principally in the retinal pigment epithelium (24), a cell layer absent in the photoentrainable ex vivo retina preparation (7).     

Inactivation of the microRNA-183/96/182 cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated “miR-183C^GT/GT,” using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.MicroRNAs (miRNAs) are small, endogenous, noncoding, regulatory RNAs and represent a newly recognized level of gene-expression regulation (1–4). miRNAs have unique expression profiles in the developing and adult retina and are involved in normal development and functions of the retina in all species studied so far (5–12). miRNAs are dysregulated in the retina of retinal degenerative mouse models, suggesting their potential involvement in retinal degeneration (13, 14). Conditional inactivation of dicer, an RNase III endonuclease required for miRNA maturation in cytosol (15), in the mouse retina resulted in alteration of retinal differentiation and optic-cup patterning, increased cell death, and disorganization of axons of retinal ganglion cells (16–19), suggesting that miRNAs are important for normal development and functions of the mammalian retina. However, in vivo functions of individual miRNAs in the retina still are largely unknown.Previously, we identified a highly conserved, intergenic, sensory organ-specific, paralogous miRNA cluster, the miR-183/96/182 cluster (hereafter, miR-183/96/182), contained within an ∼4-kb genomic segment on mouse chr6qA3.3 (8, 9). In the adult retina, miR-183/96/182 is expressed specifically in all photoreceptors and in the inner nuclear layer (8, 10). Developmentally, its expression is minimal in the embryonic retina but increases dramatically after birth and peaks in the adult retina, suggesting a role for miR-183/96/182 in maturation and normal functioning of the adult retina (8, 9). Additionally, expression of miR-183/96/182 has a diurnal pattern, suggesting a potential role in rhythmic functions of the retina (8, 9). Recently, miR-183/96/182 also was shown to be light responsive, independent of the circadian cycle (20). Targeted deletion of miR-182 alone in mouse did not result in a discernible phenotype, suggesting functional compensation by miR-183 and miR-96 (21). Point mutations of miR-96 were reported to result in progressive, nonsyndromic hearing loss in both human (22) and mouse (23); however, there was no apparent retinal phenotype, an observation that suggests miR-96 plays a major role in the inner ear but not in the retina (22–25). Finally, a recent report showed that knockdown of miR-183/96/182 in postmitotic rod photoreceptors in a miRNA-sponge transgenic mouse model resulted in increased susceptibility to light damage in the retina (26); however, no histological or functional defects of the retina were observed under normal lighting conditions (26). Thus, in vivo functions of miR-183/96/182 in the retina remain uncertain.To search for the in vivo functions of miR-183/96/182, we first dissected the genomic structure of the gene encoding miR-183/96/182 (hereafter referred to as “the miR-183/96/182 gene”) and characterized a gene-trap embryonic stem cell (ESC) clone (27–29) in which the gene-trap construct was inserted downstream of the first exon of the miR-183/96/182 gene, designated as “miR-183C^GT allele.” Using this ESC clone, we generated a mouse model, designated as “miR-183C^GT/GT,” in which the miR-183/96/182 gene is inactivated, and the β-geo cassette in the gene-trap construct reliably mirrors the endogenous expression patterns of miR-183/96/182.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Stoichiometry and specific assembly of Best ion channels

Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calcium-activated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers.Bestrophin 1 is calcium-activated chloride channel (CACC) and has been shown to express in a variety of tissues (1). In the brain, Best1 plays a crucial role in the regulation of neuronal excitability and synaptic activity by releasing gliotransmitters such as glutamate and GABA from astrocytes upon G-protein–coupled receptor (GPCR) activation (2–5). In retinal pigment epithelium (RPE) cells, Best1 plays an important role in retinal homeostasis (1), and mutations in human Best1 have been implicated in several retinal degenerative diseases including Best Macular Dystrophy (BMD) (6–12) and Retinitis Pigmentosa (13).The human bestrophin family includes three additional members; hBest2, 3, and 4 (14, 15). All four members function in heterologous cells (15–18) as anion-selective channels, whose main physiological charge carrier is chloride (15, 17, 19–22), but which also permeate glutamate and GABA (3, 4).hBest1 contains six hydrophobic segments (S1–S6), with both N and C termini residing inside the cell. Two topology models have been proposed for hBest1 (15, 23). In the first model, S1, S2, S4, and S6 traverse the membrane, whereas S3 is intracellular and S5 forms a reentrant loop from outside (15). A more recent model has S1, S2, S5, and S6 traversing the membrane and S3 and S4, although hydrophobic, remaining on the intracellular side (23).Best1 is activated by Ca²⁺ with a K_d of ∼150 nM (24). Several pieces of evidence suggest that this activation is due to direct binding of Ca²⁺ (25, 26) to an EF hand located immediately after S6 (24). It is unclear how Ca²⁺-binding gates the channel and whether the EF hand is part of the gate or communicates with it.Although much progress has been made on Best channels (15, 17, 19–22, 27), several fundamental aspects of the structure and function of this channel family are not understood. First, previous biochemical analysis has indicated that Best1 is multimeric (22, 27) but led to conflicting assessments of the number of subunits in the channel, with experiments on human Best1 suggesting a tetramer or pentamer (22) but experiments on porcine Best1 suggesting a dimer (27). Second, although coimmunoprecipitation suggests that hBest1 interacts with hBest2 (22), it is unclear if this is direct interaction. Moreover, virtually nothing is known about the determinants of assembly.In this study, we used single-molecule subunit counting and colocalization to address four major questions about the subunit assembly and function of hBest channels: (i) What is the subunit stoichiometry of hBest channels? (ii) Does hBest1 coassemble with any other member of the hBest family or with a member of different CACC family, transmembrane member 16A (TMEM16A)? (iii) How is subunit assembly specified? (iv) Does the assembly determinant play any role in channel function?     

ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.Familial exudative vitreoretinopathy (FEVR) is a well-characterized developmental anomaly of the retinal vasculature that was first described by Criswick and Schepens in 1969 (1). The most prominent characteristics of the disease result from the incomplete and aberrant vascularization of the peripheral retina and/or retinal blood vessel differentiation (2). The latter can lead to various complications, such as retinal neovascularization and exudates, vitreous hemorrhage, vitreoretinal traction, ectopia of the macula, and retinal folds and detachments (3). The clinical signs in affected individuals can be diverse, ranging from hardly detectable vascular anomalies in the peripheral retina in asymptomatic individuals to bilateral retinal detachments leading to blindness.FEVR displays all Mendelian forms of inheritance (4–8). To date, mutations in genes encoding frizzled 4 (FZD4), low-density lipoprotein receptor-related protein 5 (LRP5), tetraspanin-12 (TSPAN12), and Norrin [encoded by Norrie Disease Pseudoglioma (NDP)] have been shown to cause FEVR (4, 9–12). The proteins encoded by these genes participate in the norrin/β-catenin pathway, in which LRP5 and FZD4 act as coreceptors, NDP as a ligand for FZD4 (13) and TSPAN12 as an aid for FZD4 multimerization (14). Mutations in these genes explain up to ∼50% of all reported FEVR cases (3, 10, 11, 15).We previously excluded mutations in FZD4, LRP5, and TSPAN12 in 10 Dutch probands with adFEVR (3, 15). In the largest genetically unsolved adFEVR family, linkage analysis was combined with an unbiased exome sequencing approach of two distantly related relatives, leading to identification of a missense mutation in zinc finger protein 408 (ZNF408) as a novel cause of autosomal dominant (ad) FEVR. Cellular transfection studies suggest a dominant-negative disease mechanism, and morpholino (MO)-induced knockdown of znf408 in zebrafish show its involvement in development of the retinal vasculature.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.