Publications by authors named "Davide Comoletti"

49 Publications

Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca homeostasis and network synchrony via PMCA2/ATP2B2.

Neuron 2021 Dec 16. Epub 2021 Dec 16.

Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Autism and Neurodevelopment, Northwestern University, Chicago, IL 60611, USA; Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca homeostasis and neuronal network synchrony by a shed ectodomain.
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http://dx.doi.org/10.1016/j.neuron.2021.11.025DOI Listing
December 2021

In trans neuregulin3-Caspr3 interaction controls DA axonal bassoon cluster development.

Curr Biol 2021 08 17;31(15):3330-3342.e7. Epub 2021 Jun 17.

Department of Neurosciences, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106, USA. Electronic address:

Dopamine (DA) transmission is critical to motivation, movement, and emotion. Unlike glutamatergic and GABAergic synapses, the development of DA synapses is less understood. We show that bassoon (BSN) clusters along DA axons in the core of nucleus accumbens (NAcc) were increased in neonatal stages and reduced afterward, suggesting DA synapse elimination. Remarkably, DA neuron-specific ablating neuregulin 3 (NRG3), a protein whose levels correlate with BSN clusters, increased the clusters and impaired DA release and behaviors related to DA transmission. An unbiased screen of transmembrane proteins with the extracellular domain (ECD) of NRG3 identified Caspr3 (contactin associate-like protein 3) as a binding partner. Caspr3 was enriched in striatal medium spiny neurons (MSNs). NRG3 and Caspr3 interact in trans, which was blocked by Caspr3-ECD. Caspr3 null mice displayed phenotypes similar to those in DAT-Nrg3 mice in DA axonal BSN clusters and DA transmission. Finally, in vivo disruption of the NRG3-Caspr3 interaction increased BSN clusters. Together, these results demonstrate that DA synapse development is controlled by trans interaction between NRG3 in DA neurons and Caspr3 in MSNs, identifying a novel pair of cell adhesion molecules for brain circuit wiring.
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http://dx.doi.org/10.1016/j.cub.2021.05.045DOI Listing
August 2021

The structure-function relationship of a signaling-competent, dimeric Reelin fragment.

Structure 2021 10 4;29(10):1156-1170.e6. Epub 2021 Jun 4.

Child Health Institute of New Jersey, New Brunswick, NJ 08901, USA; Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand. Electronic address:

Reelin operates through canonical and non-canonical pathways that mediate several aspects of brain development and function. Reelin's dimeric central fragment (CF), generated through proteolytic cleavage, is required for the lipoprotein-receptor-dependent canonical pathway activation. Here, we analyze the signaling properties of a variety of Reelin fragments and measure the differential binding affinities of monomeric and dimeric CF fragments to lipoprotein receptors to investigate the mode of canonical signal activation. We also present the cryoelectron tomography-solved dimeric structure of Reelin CF and support it using several other biophysical techniques. Our findings suggest that Reelin CF forms a covalent parallel dimer with some degree of flexibility between the two protein chains. As a result of this conformation, Reelin binds to lipoprotein receptors in a manner inaccessible to its monomeric form and is capable of stimulating canonical pathway signaling.
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http://dx.doi.org/10.1016/j.str.2021.05.012DOI Listing
October 2021

Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules.

Neuropharmacology 2021 02 6;184:108381. Epub 2020 Nov 6.

Lab 'Architecture et Fonction des Macromolécules Biologiques (AFMB)', Centre National de la Recherche Scientifique (CNRS)/Aix-Marseille Univ, Faculté des Sciences - Campus Luminy, Marseille, France. Electronic address:

Cell adhesion generally involves formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/β-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all animal phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.
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http://dx.doi.org/10.1016/j.neuropharm.2020.108381DOI Listing
February 2021

Purification of a heterodimeric Reelin construct to investigate binding stoichiometry.

Eur Biophys J 2020 Dec 14;49(8):773-779. Epub 2020 Oct 14.

Child Health Institute of New Jersey, New Brunswick, NJ, 08901, USA.

Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein's function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin's central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin's known receptors, calculating the K of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.
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http://dx.doi.org/10.1007/s00249-020-01465-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7701066PMC
December 2020

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer.

Nat Commun 2020 10 14;11(1):5171. Epub 2020 Oct 14.

VIB Center for Brain & Disease Research, Herestraat 49, 3000, Leuven, Belgium.

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.
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http://dx.doi.org/10.1038/s41467-020-18956-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560607PMC
October 2020

In utero exposure to endogenous maternal polyclonal anti-Caspr2 antibody leads to behavioral abnormalities resembling autism spectrum disorder in male mice.

Sci Rep 2020 09 2;10(1):14446. Epub 2020 Sep 2.

Center for Autoimmune Musculoskeletal and Hematopoietic Diseases, Institute of Molecular Medicine, Feinstein Institutes for Medical Research, 350 Community Drive, Manhasset, NY, 11030, USA.

The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.
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http://dx.doi.org/10.1038/s41598-020-71201-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468145PMC
September 2020

Inhaled modified angiotensin converting enzyme 2 (ACE2) as a decoy to mitigate SARS-CoV-2 infection.

N Z Med J 2020 05 22;133(1515):112-118. Epub 2020 May 22.

Department of Clinical Immunology, Auckland Hospital, Auckland.

COVID-19 is a new zoonotic disease caused by the SARS-CoV-2 virus. Since its emergence in Wuhan City, China, the virus has rapidly spread across the globe causing calamitous health, economic and societal consequences. It causes disproportionately severe disease in the elderly and those with co-morbidities, such as hypertension and diabetes. There is currently no proven treatment for COVID-19 and a safe and effective vaccine is at least a year away. The virus gains access to the respiratory epithelium through cell surface angiotensin converting enzyme 2 (ACE2). The receptor binding domain (RBD) of the virus is unlikely to mutate without loss of pathogenicity and thus represents an attractive target for antiviral treatment. Inhaled modified recombinant human ACE2, may bind SARS-CoV-2 and mitigate lung damage. This decoy strategy is unlikely to provoke an adverse immune response and may reduce morbidity and mortality in high-risk groups.
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May 2020

Case Report: Is Catatonia a Clinical Feature of the Natural Progression of NLGN2-Related Neurodevelopmental Disorder?

J Autism Dev Disord 2021 01;51(1):371-376

Cincinnati Children's Hospital Medical Center Department of Human Genetics, 3333 Burnet Rd, Cincinnati, OH, 45429, USA.

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http://dx.doi.org/10.1007/s10803-020-04531-2DOI Listing
January 2021

A Proteomic Screen of Neuronal Cell-Surface Molecules Reveals IgLONs as Structurally Conserved Interaction Modules at the Synapse.

Structure 2019 06 4;27(6):893-906.e9. Epub 2019 Apr 4.

Child Health Institute of New Jersey, New Brunswick, NJ 08901, USA; Departments of Neuroscience and Cell Biology Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand. Electronic address:

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft.
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http://dx.doi.org/10.1016/j.str.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609445PMC
June 2019

An ELISA-Based Screening Platform for Ligand-Receptor Discovery.

Methods Enzymol 2019 28;615:453-475. Epub 2018 Dec 28.

Child Health Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States. Electronic address:

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.
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http://dx.doi.org/10.1016/bs.mie.2018.10.001DOI Listing
November 2019

Secreted amyloid-β precursor protein functions as a GABAR1a ligand to modulate synaptic transmission.

Science 2019 01;363(6423)

VIB Center for Brain & Disease Research, Leuven, Belgium.

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABAR1a). sAPP-GABAR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABAR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABAR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABAR1a function to modulate synaptic transmission.
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http://dx.doi.org/10.1126/science.aao4827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366617PMC
January 2019

TCRα-TCRβ pairing controls recognition of CD1d and directs the development of adipose NKT cells.

Nat Immunol 2017 01 21;18(1):36-44. Epub 2016 Nov 21.

Child Health Institute of New Jersey, Rutgers University, New Brunswick, New Jersey, USA.

The interaction between the T cell antigen receptor (TCR) expressed by natural killer T cells (NKT cells) and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC). Our molecular modeling suggested that a hydrophobic patch created after TCRα-TCRβ pairing has a role in maintaining the conformation of the NKT cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells.
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http://dx.doi.org/10.1038/ni.3622DOI Listing
January 2017

Structural Insights into Reelin Function: Present and Future.

Front Cell Neurosci 2016 27;10:137. Epub 2016 May 27.

Child Health Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers UniversityNew Brunswick, NJ, USA; Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers UniversityNew Brunswick, NJ, USA; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers UniversityNew Brunswick, NJ, USA.

Reelin is a neuronal glycoprotein secreted by the Cajal-Retzius cells in marginal regions of the cerebral cortex and the hippocampus where it plays important roles in the control of neuronal migration and the formation of cellular layers during brain development. This 3461 residue-long protein is composed of a signal peptide, an F-spondin-like domain, eight Reelin repeats (RR1-8), and a positively charged sequence at the C-terminus. Biochemical data indicate that the central region of Reelin binds to the low-density lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and the very-low-density lipoprotein receptor (VLDLR), leading to the phosphorylation of the intracellular adaptor protein Dab1. After secretion, Reelin is rapidly degraded in three major fragments, but the functional significance of this degradation is poorly understood. Probably due to its large mass and the complexity of its architecture, the high-resolution, three-dimensional structure of Reelin has never been determined. However, the crystal structures of some of the RRs have been solved, providing important insights into their fold and the interaction with the ApoER2 receptor. This review discusses the current findings on the structure of Reelin and its binding to the ApoER2 and VLDLR receptors, and we discuss some areas where proteomics and structural biology can help understanding Reelin function in brain development and human health.
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http://dx.doi.org/10.3389/fncel.2016.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882317PMC
June 2016

Super-complexes of adhesion GPCRs and neural guidance receptors.

Nat Commun 2016 Apr 19;7:11184. Epub 2016 Apr 19.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK.

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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http://dx.doi.org/10.1038/ncomms11184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838878PMC
April 2016

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1.

J Biol Chem 2016 Mar 31;291(11):5788-5802. Epub 2015 Dec 31.

From the Child Health Institute of New Jersey and Departments of Neuroscience and Cell Biology and; Pediatrics, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, New Jersey 08901,. Electronic address:

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.
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http://dx.doi.org/10.1074/jbc.M115.705681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786715PMC
March 2016

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

Biochem J 2016 Feb 30;473(4):423-34. Epub 2015 Nov 30.

Department of Biology and Biotechnology "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.
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http://dx.doi.org/10.1042/BJ20150274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747159PMC
February 2016

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development.

Structure 2015 Sep 30;23(9):1665-1677. Epub 2015 Jul 30.

Child Health Institute of New Jersey and Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers University, 89 French Street, New Brunswick, NJ 08901, USA; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ 08901, USA. Electronic address:

Latrophilins (LPHNs) are adhesion-like G-protein-coupled receptors implicated in attention-deficit/hyperactivity disorder. Recently, LPHN3 was found to regulate excitatory synapse number through trans interactions with fibronectin leucine-rich repeat transmembrane 3 (FLRT3). By isothermal titration calorimetry, we determined that only the olfactomedin (OLF) domain of LPHN3 is necessary for FLRT3 association. By multi-crystal native single-wavelength anomalous diffraction phasing, we determined the crystal structure of the OLF domain. This structure is a five-bladed β propeller with a Ca(2+) ion bound in the central pore, which is capped by a mobile loop that allows the ion to exchange with the solvent. The crystal structure of the OLF/FLRT3 complex shows that LPHN3-OLF in the closed state binds with high affinity to the concave face of FLRT3-LRR with a combination of hydrophobic and charged residues. Our study provides structural and functional insights into the molecular mechanism underlying the contribution of LPHN3/FLRT3 to the development of glutamatergic synapses.
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http://dx.doi.org/10.1016/j.str.2015.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930246PMC
September 2015

LRP8-Reelin-Regulated Neuronal Enhancer Signature Underlying Learning and Memory Formation.

Neuron 2015 May 16;86(3):696-710. Epub 2015 Apr 16.

Howard Hughes Medical Institute, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

One of the exceptional properties of the brain is its ability to acquire new knowledge through learning and to store that information through memory. The epigenetic mechanisms linking changes in neuronal transcriptional programs to behavioral plasticity remain largely unknown. Here, we identify the epigenetic signature of the neuronal enhancers required for transcriptional regulation of synaptic plasticity genes during memory formation, linking this to Reelin signaling. The binding of Reelin to its receptor, LRP8, triggers activation of this cohort of LRP8-Reelin-regulated neuronal (LRN) enhancers that serve as the ultimate convergence point of a novel synapse-to-nucleus pathway. Reelin simultaneously regulates NMDA-receptor transmission, which reciprocally permits the required γ-secretase-dependent cleavage of LRP8, revealing an unprecedented role for its intracellular domain in the regulation of synaptically generated signals. These results uncover an in vivo enhancer code serving as a critical molecular component of cognition and relevant to psychiatric disorders linked to defects in Reelin signaling.
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http://dx.doi.org/10.1016/j.neuron.2015.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486257PMC
May 2015

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis.

Nat Protoc 2014 Sep 7;9(9):2061-74. Epub 2014 Aug 7.

Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.

Ligand-receptor interactions represent essential biological triggers that regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol that couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared with previous approaches, our analysis increases sensitivity, shortens analysis duration and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These 'ecto-Fcs' are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity-purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion-trap mass spectrometers. In 4 working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our 'ecto-Fc MS' approach outperforms antibody-based approaches and provides a reproducible and robust framework for identifying extracellular ligand-receptor interactions.
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http://dx.doi.org/10.1038/nprot.2014.140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384169PMC
September 2014

Reelin induces Erk1/2 signaling in cortical neurons through a non-canonical pathway.

J Biol Chem 2014 Jul 29;289(29):20307-17. Epub 2014 May 29.

From the Department of Cell Biology and Neuroscience, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854,

Reelin is an extracellular protein that controls many aspects of pre- and postnatal brain development and function. The molecular mechanisms that mediate postnatal activities of Reelin are not well understood. Here, we first set out to express and purify the full length Reelin protein and a biologically active central fragment. Second, we investigated in detail the signal transduction mechanisms elicited by these purified Reelin proteins in cortical neurons. Unexpectedly, we discovered that the full-length Reelin moiety, but not the central fragment, is capable of activating Erk1/2 signaling, leading to increased p90RSK phosphorylation and the induction of immediate-early gene expression. Remarkably, Erk1/2 activation is not mediated by the canonical signal transduction pathway, involving ApoER2/VLDLR and Dab1, that mediates other functions of Reelin in early brain development. The activation of Erk1/2 signaling likely contributes to the modulation of neuronal maturation and synaptic plasticity by Reelin in the postnatal and adult brain.
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http://dx.doi.org/10.1074/jbc.M114.576249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106344PMC
July 2014

LPHN3, a presynaptic adhesion-GPCR implicated in ADHD, regulates the strength of neocortical layer 2/3 synaptic input to layer 5.

Neural Dev 2014 Apr 17;9. Epub 2014 Apr 17.

Neurobiology Section, Division of Biology, University of California San Diego, La Jolla, CA 92093, USA.

Background: Latrophilins (LPHNs) are a small family of neuronal adhesion-GPCRs originally discovered as receptors for the black widow spider toxin α-latrotoxin. Mutations in LPHN3 have recently been identified as risk factors for attention deficit hyperactivity disorder (ADHD) in humans, but their physiological function has remained elusive. In this study, we tested two hypotheses regarding LPHN3 function: (1) LPHN3 regulates synaptic transmission by modulating probability of release; and (2) LPHN3 controls synapse development and the abundance of synapses.

Results: We manipulated LPHN3 expression in mouse layer 2/3 (L2/3) pyramidal neurons and examined the consequences on the L2/3 to L5 cortical microcircuit. Employing an optogenetic strategy combined with shRNA knockdown of LPHN3, we found that LPHN3 did not influence probability of release at synapses formed by L2/3 neurons onto L5 pyramidal cells. The strength of L2/3 afferent input to L5, however, was weakened by loss of LPHN3. Using Synaptophysin-GFP as an anatomical marker of presynaptic terminals, we found that the density of synapses formed by L2/3 axons in L5 was reduced when LPHN3 was lost. Finally, we investigated the structural organization of the extracellular domain of LPHN3. We used single particle negative stain electron microscopy to image the extracellular domain of LPHN3 and showed that the Olfactomedin and Lectin domains form a globular domain on an elongated stalk. Cell-based binding experiments with mutant proteins revealed that the Olfactomedin domain was required for binding to FLRT3, whereas both the Olfactomedin and Lectin domains were involved in binding to Teneurin 1. Mutant LPHN3 lacking the Olfactomedin domain was not capable of rescuing the deficit in presynaptic density following knockdown of endogenous LPHN3.

Conclusions: We find that LPHN3 regulates the number of synapses formed by L2/3 neurons in L5 and the strength of synaptic drive from the L2/3-L5 pathway. The Olfactomedin domain of LPHN3 is required for this effect on synapse number and binding to its postsynaptic ligand FLRT3. We propose that LPHN3 functions in synaptic development and is important in determining the connectivity rates between principal neurons in the cortex.
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http://dx.doi.org/10.1186/1749-8104-9-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996519PMC
April 2014

Cholinesterase confabs and cousins: approaching forty years.

Chem Biol Interact 2013 Mar 16;203(1):10-3. Epub 2012 Oct 16.

Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, Mail Code 0657, La Jolla, CA 92093, United States.

In the past four decades of cholinesterase (ChE) research, we have seen substantive evolution of the field from one centered around substrate and inhibitor kinetic profiles and compound characterizations to the analysis of ChE structure, first through the gene families and then by X-ray crystallographic determinations of the free enzymes and their complexes and conjugates. Indeed, these endeavors have been facilitated by recombinant DNA technologies, structure determinations and parallel studies in related proteins in the α/β-hydrolase fold family. This approach has not only contributed to a fundamental understanding of structure and function of a large family of hydrolase-like proteins possessing functions other than catalysis, but also has been used to develop new practical strategies for scavenging and antidotal activity in cases of organophosphate insecticide or nerve agent exposure.
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http://dx.doi.org/10.1016/j.cbi.2012.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618580PMC
March 2013

Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation.

Hum Mol Genet 2012 Nov 7;21(21):4761-73. Epub 2012 Aug 7.

Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA.

Although genetic variations in several genes encoding for synaptic adhesion proteins have been found to be associated with autism spectrum disorders, one of the most consistently replicated genes has been CNTNAP2, encoding for contactin-associated protein-like 2 (CASPR2), a multidomain transmembrane protein of the neurexin superfamily. Using immunofluorescence confocal microscopy and complementary biochemical techniques, we compared wild-type CASPR2 to 12 point mutations identified in individuals with autism. In contrast to the wild-type protein, localized to the cell surface, some of the mutants show altered cellular disposition. In particular, CASPR2-D1129H is largely retained in the endoplasmic reticulum (ER) in HEK-293 cells and in hippocampal neurons. BiP/Grp78, Calnexin and ERp57, key ER chaperones, appear to be responsible for retention of this mutant and activation of one signaling pathway of the unfolded protein response (UPR). The presence of this mutation also lowers expression and activates proteosomal degradation. A frame-shift mutation that causes a form of syndromic epilepsy (CASPR2-1253*), results in a secreted protein with seemingly normal folding and oligomerization. Taken together, these data indicate that CASPR2-D1129H has severe trafficking abnormalities and CASPR2-1253* is a secreted soluble protein, suggesting that the structural or signaling functions of the membrane tethered form are lost. Our data support a complex genetic architecture in which multiple distinct risk factors interact with others to shape autism risk and presentation.
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http://dx.doi.org/10.1093/hmg/dds320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471401PMC
November 2012

Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic β cell function.

J Biol Chem 2012 Jun 23;287(24):19816-26. Epub 2012 Apr 23.

Department of Medicine and Pediatric Diabetes Research Center, UCSD School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring β cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into β cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that β cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the β cell surface and contributes to the increased insulin secretion brought about by β cell-to-β cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 β cell proliferation and insulin content. These results extend the known parallels between the synaptic and β cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of β cell function.
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http://dx.doi.org/10.1074/jbc.M111.280537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370167PMC
June 2012

FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.

Neuron 2012 Mar;73(5):903-10

Neurobiology Section, Division of Biology, University of California San Diego, La Jolla, CA 92093, USA.

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.
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http://dx.doi.org/10.1016/j.neuron.2012.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326387PMC
March 2012

Processing of cholinesterase-like α/β-hydrolase fold proteins: alterations associated with congenital disorders.

Protein Pept Lett 2012 Feb;19(2):173-9

Dipartimento di Biologia e Biotecnologie "Charles Darwin", Universita' di Roma "La Sapienza", Italy.

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.
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http://dx.doi.org/10.2174/092986612799080103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755491PMC
February 2012

The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function.

Structure 2011 Jun 27;19(6):767-78. Epub 2011 May 27.

Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

α- and β-neurexins (NRXNs) are transmembrane cell adhesion proteins that localize to presynaptic membranes in neurons and interact with the postsynaptic neuroligins (NLGNs). Their gene mutations are associated with the autism spectrum disorders. The extracellular region of α-NRXNs, containing nine independently folded domains, has structural complexity and unique functional characteristics, distinguishing it from the smaller β-NRXNs. We have solved the X-ray crystal structure of seven contiguous domains of the α-NRXN-1 extracellular region at 3.0 Å resolution. The structure reveals an arrangement where the N-terminal five domains adopt a more rigid linear conformation and the two C-terminal domains form a separate arm connected by a flexible hinge. In an extended conformation the molecule is suitably configured to accommodate a bound NLGN molecule, as supported by structural comparison and surface plasmon resonance. These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1.
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http://dx.doi.org/10.1016/j.str.2011.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279696PMC
June 2011

The macromolecular architecture of extracellular domain of alphaNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition.

Structure 2010 Aug;18(8):1044-53

Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Neurexins are multidomain synaptic cell-adhesion proteins that associate with multiple partnering proteins. Genetic evidence indicates that neurexins may contribute to autism, schizophrenia, and nicotine dependence. Using analytical ultracentrifugation, single-particle electron microscopy, and solution X-ray scattering, we obtained a three-dimensional structural model of the entire extracellular domain of neurexin-1alpha. This protein adopts a dimensionally asymmetric conformation that is monomeric in solution, with a maximum dimension of approximately 170 A. The extracellular domain of alpha-neurexin maintains a characteristic "Y" shape, whereby LNS domains 1-4 form an extended base of the "Y" and LNS5-6 the shorter arms. Moreover, two major regions of flexibility are present: one between EGF1 and LNS2, corresponding to splice site 1, another between LNS5 and 6. We thus provide the first structural insights into the architecture of the extracellular region of neurexin-1alpha, show how the protein may fit in the synaptic cleft, and how partnering proteins could bind simultaneously.
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http://dx.doi.org/10.1016/j.str.2010.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948785PMC
August 2010
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