The evolution of a nervous system as a control system of the body's functions is a key innovation of animals. Its fundamental units are neurons, highly specialized cells dedicated to fast cell-cell ...communication. Neurons pass signals to other neurons, muscle cells, or gland cells at specialized junctions, the synapses, where transmitters are released from vesicles in a Ca
2+
-dependent fashion to activate receptors in the membrane of the target cell. Reconstructing the origins of neuronal communication out of a more simple process remains a central challenge in biology. Recent genomic comparisons have revealed that all animals, including the nerveless poriferans and placozoans, share a basic set of genes for neuronal communication. This suggests that the first animal, the Urmetazoan, was already endowed with neurosecretory cells that probably started to connect into neuronal networks soon afterward. Here, we discuss scenarios for this pivotal transition in animal evolution.
Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal ...morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites.
Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans-one of the ...early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.
In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and ...transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.
Neuroligins (NL1-NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare ...forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimuluscoding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.
For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as ...synaptosome preparations contain multiple types of synapses and non‐synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high‐resolution biochemical analyses of specific synapse subpopulations. Employing knock‐in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.
Synopsis
This study describes the establishment, validation, and application of a new fluorescence‐activated sorting method ‐ Fluorescence Activated Synaptosome Sorting or FASS ‐ that allows for purification of glutamatergic synaptosomes from knock‐in mutant mice expressing the fluorescent synaptic marker protein VGLUT1‐Venus.
Fluorescence Activated Synaptosome Sorting from samples of VGLUT1‐Venus knock‐in mice enriches glutamatergic synaptosomes to near homogeneity.
Glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting contain bona fide glutamatergic synapse proteins but lack other synapse types and glia cell contaminants.
Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting allows for identifying and cataloguing known and novel components of glutamatergic synapses.
Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting identified FXYD6 and Tpd52 as novel components of glutamatergic synapses.
A novel Fluorescence Activated Synaptosome Sorting (FASS) approach allows for high‐resolution biochemical analyses of specific synapse subpopulations and identification of 163 proteins that are specifically enriched in FASS‐sorted synaptosomes.
The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory ...GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin‐based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin‐2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans‐synaptic neuroligin‐dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin‐based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.
Synopsis
The postsynaptic cell adhesion protein neuroligin‐2 stabilizes the open/active conformation of collybistin to regulate gephyrin and GABAA receptor clustering at postsynaptic plasma membranes.
Collybistin regulates the recruitment of the postsynaptic scaffold protein gephyrin to inhibitory synapses in the brain, which, in turn, leads to the postsynaptic clustering of GABAA (and glycine) receptors.
Collybistin exhibits a dynamic equilibrium between closed/inactive and open/active conformations and requires a conformational activation for its phosphoinositide‐binding and gephyrin‐clustering activity.
At synapses, the active conformation of collybistin is selected by interactions with the postsynaptic cell adhesion proteins neuroligin‐2 and neuroligin‐4 and is further stabilized by the subsequent formation of a neuroligin/gephyrin/collybistin holocomplex.
Structural and functional data show that the adhesion protein neuroligin‐2 stabilizes the open, active conformation of the adaptor collybistin, thus regulating gephyrin and GABAA receptor clustering at postsynaptic membranes.
Autism spectrum conditions (ASCs) are heritable conditions characterized by impaired reciprocal social interactions, deficits in language acquisition, and repetitive and restricted behaviors and ...interests. In addition to more complex genetic susceptibilities, even mutation of a single gene can lead to ASC. Several such monogenic heritable ASC forms are caused by loss-of-function mutations in genes encoding regulators of synapse function in neurons, including NLGN4. We report that mice with a loss-of-function mutation in the murine NLGN4 ortholog Nlgn4, which encodes the synaptic cell adhesion protein Neuroligin-4, exhibit highly selective deficits in reciprocal social interactions and communication that are reminiscent of ASCs in humans. Our findings indicate that a protein network that regulates the maturation and function of synapses in the brain is at the core of a major ASC susceptibility pathway, and establish Neuroligin-4-deficient mice as genetic models for the exploration of the complex neurobiological disorders in ASCs.
The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action ...potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.
Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the ...strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca
2+ levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca
2+ triggered release of transmitters.
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