David C. Martinelli

Presynaptic Neurexin-3 Alternative Splicing trans-Synaptically Controls Postsynaptic AMPA Receptor Trafficking

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 genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-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.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G Protein–Coupled Receptors

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.

Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog

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 cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins

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.

The role of Gas1 in embryonic development and its implications for human disease.

Growth Arrest Specific Gene 1 (Gas1) has long been regarded as a cell cycle inhibitor of the G0 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.

A Sonic Hedgehog Missense Mutation Associated with Holoprosencephaly Causes Defective Binding to GAS1

Holoprosencephaly (HPE) is a common birth defect predominantly affecting the forebrain and face and has been linked to mutations in the sonic hedgehog (SHH) gene. HPE is genetically heterogeneous, and clinical presentation represents a spectrum of phenotypes. We have previously shown that Gas1 encodes a cell-autonomous Hedgehog signaling enhancer. Combining cell surface binding, in vitro activity, and explant culture assays, we provide evidence that SHH contains a previously unknown unique binding surface for its interaction with GAS1 and that this surface is also important for maximal signaling activity. Within this surface, the Asn-115 residue of human SHH has been documented to associate with HPE when mutated to lysine (N115K). We provide evidence that HPE associated with this mutation can be mechanistically explained by a severely reduced binding of SHH to GAS1, and we predict a similar result if a mutation were to occur at Tyr-80. Our data should encourage future searches for mutations in GAS1 as possible modifiers contributing to the wide spectrum of HPE.

Gas1 is a receptor for sonic hedgehog to repel enteric axons

Significance The enteric nervous system is the largest peripheral nervous subsystem. Enteric axons form a tubular meshwork arborizing the smooth muscles of the digestive tract to control gut movement. Deficiencies of enteric neurons cause megacolon and constipation—that is, Hirschsprung’s disease. How enteric axons are kept in the smooth muscle layers without projecting toward the gut epithelium is unknown. We provide, to our knowledge, the first insights into how these axons are confined to the periphery and, in the process, define a novel mechanism of sonic hedgehog signaling for axon repulsion involving the guidance receptor growth arrest specific gene 1 and the signaling G protein α z. We believe this to be a conceptual advance in understanding a key topological problem in axonal arborization and to have important health implications. 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.

Structures of C1q-like Proteins Reveal Unique Features among the C1q/TNF Superfamily.

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.

Expression of C1ql3 in Discrete Neuronal Populations Controls Efferent Synapse Numbers and Diverse Behaviors

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.

Cerebellins meet neurexins (Commentary on Matsuda & Yuzaki)

How synapses are formed and maintained is a fundamental question in neuroscience that is assuming increasing importance for our understanding of numerous neurological disorders. Recently, proteins containing complement factor C1Q-related domains (C1q-domains) have been attracting interest because of their potential role in synapse formation and ⁄ or maintenance. C1q-domains are small globular domains found in the eponymous complement factor C1Q and in a multitude of other proteins, including both small secreted proteins such as cerebellins and C1ql proteins, and large multidomain proteins such as emilins and multimerins (Ghai et al., 2007). Accumulating evidence is now implicating several C1q-domain proteins in synapse formation and ⁄ or elimination: cerebellin-1 was revealed to function in synapse maturation (Yuzaki, 2009) (Hirai et al., 2005), complement factor C1Q was implicated in synapse elimination (Stevens et al., 2007), and members of a third C1q-domain protein family, C1qls (for C1q-like), were demonstrated to reduce synapse numbers in cultured neurons (Bolliger et al., 2011). New data now published in this issue of EJN (Matsuda and Yuzaki, 2011), complementing earlier reports published in Cell and Science (Matsuda et al., 2010; Uemura et al., 2010), reveal that cerebellin-1 functions in synapse formation by binding to the presynaptic cell-adhesion neurexin molecules, and that other cerebellin isoforms may also do so. Cerebellin-1 is secreted from cerebellar granule cells, and acts as a ligand for the postsynaptic glutamate receptor (GluR)d2 on Purkinje cells (Matsuda et al., 2010). Cerebellin-1 additionally binds to presynaptic neurexin-1b, resulting in a trimeric trans-synaptic complex composed of cerebellin-1, GluRd2, and neurexin-1b (Uemura et al., 2010). In the present EJN article, Matsuda and Yuzaki use cell-based binding assays to demonstrate that cerebellin-1 also binds to neurexin-1a, as well as to all three b-neurexins. This binding interaction requires the inclusion of an alternatively spliced exon known as ‘splice site #4’ (Ushkaryov et al., 1992), and appears to be Ca-independent. The cerebellin-1 binding properties for neurexins are thus distinct from those of neuroligins (Ichtchenko et al., 1995) or LRRTMs (Ko et al., 2009; de Wit et al., 2009). This result suggests that alternative splicing of neurexins at splice site #4 influences whether they bind to either cerebellin-1 or to neuroligins and LRRTM2. Matsuda and Yuzaki further demonstrate that cerebellin-2 (but, interestingly, not cerebellin-4) also binds to neurexin-1b. The authors provide evidence that cerebellin-1 and cerebellin-2 have similar synaptogenic activities in cultured hippocampal and cortical neurons, not just in cerebellar neurons. Thus, the new article provides further evidence that cerebellin-1 acts as a bi-directional synapse organizer, initiating the recruitment of both presynaptic and postsynaptic proteins to the points of contact with both neurexin-1b and GluRd2. The picture of cerebellins emerging from these studies suggests that they function as ‘connectors’, linking presynaptic neurexins to postsynaptic GluRd-type receptors, and that, in doing so, they activate synapse formation. This attractive hypothesis raises a slew of new questions. For example, what is the mechanism by which such binding activates synapse formation – possibly by multimerization of the binding partners – and does this binding initiate synapse formation, or stabilize transient synapses established by other mechanisms? On a higher level, these results raise the questions of whether cerebellin binding is the primary function of neurexin proteins containing splice site #4, how important such binding is for the formation of brain circuits, and whether other postsynaptic cerebellin receptors exist. Answers to these questions will yield a greater understanding of how synapses are formed and maintained in the cerebellum and the numerous other brain regions where cerebellin family members are expressed.