Women Breaking Ground: A Website Profiling Women Entrepreneurs in Syracuse

My Honors Capstone project is a website titled Women Breaking Ground (www.womenbreakingground.com). On my website, I published 10 feature-length profiles on women entrepreneurs in Syracuse. Each profile features photographs and an interactive map to show readers the location of the woman-owned business. This project is relevant because the number of women business owners in the United States is increasing; in fact, women are opening 1,200 new businesses every day. I hope that my project inspires even more women to pursue their passion and become entrepreneurs

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

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

Neurolign 3 misfolding mutations and activation of the unfolded protein response

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.This work was supported by: Compagnia San Paolo, Sapienza University of Rome and Pasteur Institute - Cenci Bolognetti Foundation grants to ADJ. SJM is a MRC Senior Clinical Research Fellow [MRC Ref G1002610]. This work was also supported by National Institutes of Health grants (MH092906), from the Robert Wood Johnson Foundation to the Child Health Institute of New Jersey [grant #67038] and to the Governor's Council for Medical Research and Treatment of Autism [CAUT14APL028] to D.C.This is the final version of the article. It first appeared from Portland Press via https://doi.org/http://dx.doi.org/10.1042/BJ2015027

Trafficking of cholinesterases and neuroligins mutant proteins. An association with autism

Autism encompasses a wide spectrum of disorders arising during brain development. Recent studies reported that sequence polymorphisms in neuroligin-3 (NLGN3) and neuroligin-4 (NLGN4) genes have been linked to autism spectrum disorders indicating neuroligin genes as candidate targets in brain disorders. We have characterized a single mutation found in two affected brothers that substituted Arg451 to Cys in NL3. Our data show that the exposed Cys causes retention of the protein in the endoplasmic reticulum (ER) when expressed in HEK-293 cells. To examine whether the introduction of a Cys in the C-terminal region of other α/β-hydrolase fold proteins could promote the same cellular phenotype, we made homologous mutations in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and found a similar processing deficiency and intracellular retention (De Jaco et al., J Biol Chem. 2006, 281:9667-76). NL3, AChE and BChE mutant proteins are recognized as misfolded in the ER, and degraded via the proteasome pathway. A 2D electrophoresis coupled with mass spectrometry based approach was used to analyze proteins co-immunoprecipitating with NL3 and show differential expression of factors interacting with wild type and mutant NL3. We identified several proteins belonging to distinct ER resident chaperones families, including calnexin, responsible for playing a role in the folding steps of the AChE and NLs. © 2008 Elsevier Ireland Ltd. All rights reserved

Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein

Functional Phenotypic Rescue of Caenorhabditis elegans Neuroligin-Deficient Mutants by the Human and Rat NLGN1 Genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders

Computational geometry analysis of dendritic spines by structured illumination microscopy

We are currently short of methods that can extract objective parameters of dendritic spines useful for their categorization. Authors present in this study an automatic analytical pipeline for spine geometry using 3D-structured illumination microscopy, which can effectively extract many geometrical parameters of dendritic spines without bias and automatically categorize spine population based on their morphological feature

Structural Basis for Variant-Specific Neuroligin-Binding by α-Neurexin

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function

Homology modelling and spectroscopy, a never-ending love story

Homology modelling is normally the technique of choice when experimental structure data are not available but three-dimensional coordinates are needed, for example, to aid with detailed interpretation of results of spectroscopic studies. Herein, the state of the art of homology modelling will be described in the light of a series of recent developments, and an overview will be given of the problems and opportunities encountered in this field. The major topic, the accuracy and precision of homology models, will be discussed extensively due to its influence on the reliability of conclusions drawn from the combination of homology models and spectroscopic data. Three real-world examples will illustrate how both homology modelling and spectroscopy can be beneficial for (bio)medical research