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Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [10] containing a GPCR proteolysis site (GPS). The N-terminal extracellular region often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [104, 418]. Several receptors have been suggested to function as mechanosensors [320, 288, 396, 38]. Cryo-EM structures of the 7-transmembrane domain of several adhesion GPCRs have been determined recently [292, 21, 403, 212, 300, 302, 431, 293]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [125].

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [9] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [101, 403]. Several receptors have been suggested to function as mechanosensors [309, 280, 383, 35]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [122].

Hearing depends on the transduction of sounds into neural signals by the inner hair cells of the cochlea. Cochleae also have outer hair cells with unique electromotile properties that increase auditory sensitivity, but they are particularly susceptible to damage by intense noise exposure, ototoxic drugs, and aging. Although the outer hair cells have synapses on afferent neurons that project to the brain, the function of this neuronal circuit is unclear. Here, we created a novel mouse allele that inserts a fluorescent reporter at the C1ql1 locus which revealed gene expression in the outer hair cells and allowed creation of outer hair cell-specific C1ql1 knockout mice. We found that C1ql1 expression in outer hair cells corresponds to areas with the most sensitive frequencies of the mouse audiogram, and that it has an unexpected adolescence-onset developmental timing. No expression was observed in the inner hair cells. Since C1QL1 in the brain is made by neurons, transported anterogradely in axons, and functions in the synaptic cleft, C1QL1 may serve a similar function at the outer hair cell afferent synapse. Histological analyses revealed that C1ql1 conditional knockout cochleae may have reduced outer hair cell afferent synapse maintenance. However, auditory behavioral and physiological assays did not reveal a compelling phenotype. Nonetheless, this study identifies a potentially useful gene expressed in the cochlea and opens the door for future studies aimed at elucidating the function of C1QL1 and the function of the outer hair cell and its afferent neurons.

Synapses are the fundamental structural unit by which neurons communicate. An orchestra of proteins regulates diverse synaptic functions, including synapse formation, maintenance, and elimination-synapse homeostasis. Some proteins of the larger C1q super-family are synaptic organizers involved in crucial neuronal processes in various brain regions. C1Q-like (C1QL) proteins bind to the adhesion G protein-coupled receptor B3 (ADGRB3) and act at synapses in a subset of circuits. To investigate the hypothesis that the secreted C1QL proteins mediate tripartite trans-synaptic adhesion complexes, we conducted an in vivo interactome study and identified new binding candidates. We demonstrate that C1QL3 mediates a novel cell-cell adhesion complex involving ADGRB3 and two neuronal pentraxins, NPTX1 and NPTXR. Analysis of single-cell RNA-Seq data from the cerebral cortex shows that C1ql3, Nptx1, and Nptxr are highly co-expressed in the same excitatory neurons. Thus, our results suggest the possibility that in vivo the three co-expressed proteins are presynaptically secreted and form a complex capable of binding to postsynaptically localized ADGRB3, thereby creating a novel trans-synaptic adhesion complex. Identifying new binding partners for C1QL proteins and deciphering their underlying molecular principles will accelerate our understanding of their role in synapse organization.

Many biochemical, physiological, and behavioral processes such as glucose metabolism, body temperature, and sleep-wake cycles show regular daily rhythms. These circadian rhythms are adjusted to the environmental light-dark cycle by a central pacemaker located in the suprachiasmatic nucleus (SCN) in order for the processes to occur at appropriate times of day. Here, we investigated the expression and function of a synaptic organizing protein, C1QL3, in the SCN. We found that C1ql3 is robustly expressed in the SCN. C1ql3 knockout mice have a reduced density of excitatory synapses in the SCN. In addition, these mice exhibited less consolidated activity to the active portions of the day and period lengthening following a 15-minute phase-delaying light pulse. These data identify C1QL3 as a signaling molecule that is highly expressed in SCN neurons, where it contributes to the formation and/or maintenance of glutamatergic synapses and plays a role in circadian behaviors, which may include circadian aftereffects.

Summary The mammalian brain contains few niches for neural stem cells (NSCs) capable of generating new neurons, whereas other regions are primarily gliogenic. Here we leverage the spatial separation of the sub-ependymal zone NSC niche and the olfactory bulb, the region to which newly generated neurons from the sub-ependymal zone migrate and integrate, and present a comprehensive proteomic characterization of these regions in comparison to the cerebral cortex, which is not conducive to neurogenesis and integration of new neurons. We find differing compositions of regulatory extracellular matrix (ECM) components in the neurogenic niche. We further show that quiescent NSCs are the main source of their local ECM, including the multi-functional enzyme transglutaminase 2, which we show is crucial for neurogenesis. Atomic force microscopy corroborated indications from the proteomic analyses that neurogenic niches are significantly stiffer than non-neurogenic parenchyma. Together these findings provide a powerful resource for unraveling unique compositions of neurogenic niches., Graphical Abstract, Highlights • Proteomics define the NSC niche-specific extracellular matrix • Detergent-solubility profiling reveals extracellular matrix architecture • Transglutaminase 2 regulates neurogenesis • Stiffness is increased in the neurogenic niches of the brain, The physical properties of stem cell niches are thought to mediate important regulatory functions. Here we provide a proteomic resource of the neural stem cell niche in comparison to gliogenic brain parenchyma, highlighting stiffness and the enzyme transglutaminase 2 as key regulators of neurogenesis.

Cytokines and chemokines play diverse roles in different organ systems. Family with sequence similarity 19, member A1-5 (FAM19A1-A5; also known as TAFA1-5) is a group of conserved chemokine-like proteins enriched in the CNS of mice and humans. Their functions are only beginning to emerge. Here, we show that the expression of Fam19a1-a5 in different mouse brain regions are induced or suppressed by unfed and refed states. The striking nutritional regulation of Fam19a family members in the brain suggests a potential central role in regulating metabolism. Using a knockout (KO) mouse model, we show that loss of FAM19A1 results in sexually dimorphic phenotypes. In male mice, FAM19A1 deficiency alters food intake patterns during the light and dark cycle. Fam19a1 KO mice are hyperactive, and locomotor hyperactivity is more pronounced in female KO mice. Behavior tests indicate that Fam19a1 KO female mice have reduced anxiety and sensitivity to pain. Spatial learning and exploration, however, is preserved in Fam19a1 KO mice. Altered behaviors are associated with elevated norepinephrine and dopamine turnover in the striatum. Our results establish an in vivo function of FAM19A1 and highlight central roles for this family of neurokines in modulating animal physiology and behavior.-Lei, X., Liu, L., Terrillion, C. E., Karuppagounder, S. S., Cisternas, P., Lay, M., Martinelli, D. C., Aja, S., Dong, X., Pletnikov, M. V., Wong, G. W. FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors.

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [8] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [82, 332]. Several receptors have been suggested to function as mechanosensors [254, 234, 315, 32]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [100].

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

C1ql3 is a secreted neuronal protein that binds to BAI3, an adhesion-class GPCR. C1ql3 is homologous to other gC1q-domain proteins that control synapse numbers, but a role for C1ql3 in regulating synapse density has not been demonstrated. We show in cultured neurons that C1ql3 expression is activity dependent and supports excitatory synapse density. Using newly generated conditional and constitutive C1ql3 knockout mice, we found that C1ql3-deficient mice exhibited fewer excitatory synapses and diverse behavioral abnormalities, including marked impairments in fear memories. Using circuit-tracing tools and conditional ablation of C1ql3 targeted to specific brain regions, we demonstrate that C1ql3-expressing neurons in the basolateral amygdala project to the medial prefrontal cortex, that these efferents contribute to fear memory behavior, and that C1ql3 is required for formation and/or maintenance of these synapses. Our results suggest that C1ql3 is a signaling protein essential for subsets of synaptic projections and the behaviors controlled by these projections.

Holoprosencephaly (HPE) is a clinically heterogeneous developmental anomaly affecting the CNS and face, in which the embryonic forebrain fails to divide into distinct halves. Numerous genetic loci and environmental factors are implicated in HPE, but mutation in the sonic hedgehog (Shh) gene is an established cause in both humans and mice. As growth arrest-specific 1 (Gas1) encodes a membrane glycoprotein previously identified as a Shh antagonist in the somite, we analyzed the craniofacial phenotype of mice harboring a targeted Gas1 deletion. Gas1(-/-) mice exhibited microform HPE, including midfacial hypoplasia, premaxillary incisor fusion, and cleft palate, in addition to severe ear defects; however, gross integrity of the forebrain remained intact. These defects were associated with partial loss of Shh signaling in cells at a distance from the source of transcription, suggesting that Gas1 can potentiate hedgehog signaling in the early face. Loss of a single Shh allele in a Gas1(-/-) background significantly exacerbated the midline craniofacial phenotype, providing genetic evidence that Shh and Gas1 interact. As human GAS1 maps to chromosome 9q21.3-q22, a region previously associated with nonsyndromic cleft palate and congenital deafness, our results establish GAS1 as a potential locus for several human craniofacial malformations.

The myenteric plexus of the enteric nervous system controls the movement of smooth muscles in the gastrointestinal system. They extend their axons between two peripheral smooth muscle layers to form a tubular meshwork arborizing the gut wall. How a tubular axonal meshwork becomes established without invading centrally toward the gut epithelium has not been addressed. We provide evidence here that sonic hedgehog (Shh) secreted from the gut epithelium prevents central projections of enteric axons, thereby forcing their peripheral tubular distribution. Exclusion of enteric central projections by Shh requires its binding partner growth arrest specific gene 1 (Gas1) and its signaling component smoothened (Smo) in enteric neurons. Using enteric neurons differentiated from neurospheres in vitro, we show that enteric axon growth is not inhibited by Shh. Rather, when Shh is presented as a point source, enteric axons turn away from it in a Gas1-dependent manner. Of the Gαi proteins that can couple with Smo, G protein α Z (Gnaz) is found in enteric axons. Knockdown and dominant negative inhibition of Gnaz dampen the axon-repulsive response to Shh, and Gnaz mutant intestines contain centrally projected enteric axons. Together, our data uncover a previously unsuspected mechanism underlying development of centrifugal tubular organization and identify a previously unidentified effector of Shh in axon guidance.

C1q-like (C1QL) -1, -2, and -3 proteins are encoded by homologous genes that are highly expressed in brain. C1QLs bind to brain-specific angiogenesis inhibitor 3 (BAI3), an adhesion-type G-protein coupled receptor that may regulate dendritic morphology by organizing actin filaments. To begin to understand the function of C1QLs, we determined high-resolution crystal structures of the globular C1q-domains of C1QL1, C1QL2, and C1QL3. Each structure is a trimer, with each protomer forming a jelly-roll fold consisting of 10 β strands. Moreover, C1QL trimers may assemble into higher-order oligomers similar to adiponectin and contain four Ca(2+)-binding sites along the trimeric symmetry axis, as well as additional surface Ca(2+)-binding sites. Mutation of Ca(2+)-coordinating residues along the trimeric symmetry axis lowered the Ca(2+)-binding affinity and protein stability. Our results reveal unique structural features of C1QLs among C1q/TNF superfamily proteins that may be associated with their specific brain functions.

Neurexins are essential presynaptic cell-adhesion molecules that are linked to schizophrenia and autism, and are subject to extensive alternative splicing. Here, we used a novel genetic approach to test the physiological significance of neurexin alternative splicing. We generated knock-in mice in which alternatively spliced sequence #4 (SS4) of neurexin-3 is constitutively included, but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin-binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA- but not NMDA-receptor levels and enhanced postsynaptic AMPA-receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA-receptor recruitment during NMDA-receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA-receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.

C1q-like genes ( C1ql1–C1ql4 ) encode small, secreted proteins that are expressed in differential patterns in the brain but whose receptors and functions remain unknown. BAI3 protein, in contrast, is a member of the cell-adhesion class of G protein-coupled receptors that are expressed at high levels in the brain but whose ligands have thus far escaped identification. Using a biochemical approach, we show that all four C1ql proteins bind to the extracellular thrombospondin-repeat domain of BAI3 with high affinity, and that this binding is mediated by the globular C1q domains of the C1ql proteins. Moreover, we demonstrate that addition of submicromolar concentrations of C1ql proteins to cultured neurons causes a significant decrease in synapse density, and that this decrease was prevented by simultaneous addition of the thrombospondin-repeat fragment of BAI3, which binds to C1ql proteins. Our data suggest that C1ql proteins are secreted signaling molecules that bind to BAI3 and act, at least in part, to regulate synapse formation and/or maintenance.

Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.

Growth Arrest Specific Gene 1 (Gas1) has long been regarded as a cell cycle inhibitor of the G(0) to S phase transition. How GAS1, a GPI-anchored plasma membrane protein, directs intracellular changes without an extracellular ligand or a transmembrane protein partner has been puzzling. A recent series of biochemical and molecular genetic studies assigned the mammalian Hedgehog (HH) growth factor to be a ligand for GAS1 in vitro and in vivo. HH has enjoyed considerable attention for its profound role in embryonic patterning as a classic morphogen, i.e. inducing various cell types in a concentration-dependent manner. GAS1 appears to help transform the HH concentration gradient into its morphogenic activity gradient by acting cooperatively with the HH receptor, the 12-transmembrane protein Patched 1 (PTC1). These findings provoke intriguing thoughts on how HH and GAS1 may coordinate cell proliferation and differentiation to create biological patterns. The role of HH extends to human genetic diseases, stem cell renewal, and cancer growth, and we consider the possibility of GAS1's involvement in these processes as well.

Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator.