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Interaction of Acetylcholinesterase with Neurexin-1Β Regulates

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Interaction of Acetylcholinesterase with Neurexin-1Β Regulates Xiang et al. Molecular Brain 2014, 7:15 http://www.molecularbrain.com/content/7/1/15 RESEARCH Open Access Interaction of Acetylcholinesterase with Neurexin-1β regulates Glutamatergic Synaptic stability in Hippocampal neurons Yun-Yan Xiang1,2†, Haiheng Dong3†, Burton B Yang4, John F MacDonald1,2 and Wei-Yang Lu1,2,3,5* Abstract Background: Excess expression of acetylcholinesterase (AChE) in the cortex and hippocampus causes a decrease in the number of glutamatergic synapses and alters the expression of neurexin and neuroligin, trans-synaptic proteins that control synaptic stability. The molecular sequence and three-dimensional structure of AChE are homologous to the corresponding aspects of the ectodomain of neuroligin. This study investigated whether excess AChE interacts physically with neurexin to destabilize glutamatergic synapses. Results: The results showed that AChE clusters colocalized with neurexin assemblies in the neurites of hippocampal neurons and that AChE co-immunoprecipitated with neurexin from the lysate of these neurons. Moreover, when expressed in human embryonic kidney 293 cells, N-glycosylated AChE co-immunoprecipitated with non-O–glycosylated neurexin-1β,withN-glycosylation of the AChE being required for this co-precipitation to occur. Increasing extracellular AChE decreased the association of neurexin with neuroligin and inhibited neuroligin-induced synaptogenesis. The number and activity of excitatory synapses in cultured hippocampal neurons were reduced by extracellular catalytically inactive AChE. Conclusions: Excessive glycosylated AChE could competitively disrupt a subset of the neurexin–neuroligin junctions consequently impairing the integrity of glutamatergic synapses. This might serve a molecular mechanism of excessive AChE induced neurodegeneration. Keywords: Protein interaction, Glycosylation, Neurodegeneration, Synaptic apoptosis Introduction globular monomers and dimers of AChE-S. Membrane- As the key enzyme that hydrolyzes acetylcholine, acetyl- bound AChE consists of AChE-S tetramers tethered to the cholinesterase (AChE) plays a critical role in regulating cell membrane by a proline-rich membrane anchor [2]. cholinergic signaling. Neurons in the central nervous Interestingly, membrane-bound AChE-S can be released system generate two isoforms of AChE: synaptic AChE in response to cholinergic activation [3]. Collective data (AChE-S, also known as “tailed AChE”) and read-through imply that the isoforms, concentrations and localization of AChE (AChE-R). In adults, AChE-S is the predominant AChE within the brain are dynamically regulated. isoform, although AChE-R increases following exposure In the brain, AChE is produced by cholinergic neurons to a variety of stressors [1]. In the extracellular space, [4], cholinoceptive neurons [5], and astrocytes [6]. Clinical AChE exists in both soluble and membrane-bound forms. studies have indicated that an increase in anomalous Soluble AChE includes monomeric AChE-R, as well as AChE is strongly correlated with the pathogenesis of Alzheimer disease (AD) [7-10]. Specifically, AChE is a major component of amyloid-β (Aβ) plaques [11], and * Correspondence: [email protected] N- †Equal contributors glycosylated AChE is increased in the cerebrospinal 1Robarts Research Institute, University of Western Ontario, London, Ontario, fluid of patients with AD [12]. In particular, AChE binds Canada to Aβ, thus promoting both the formation of Aβ fibrils 2Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada [13] and the occurrence of neurotoxicity [14]. Moreover, Full list of author information is available at the end of the article augmenting AChE expression in the brains of transgenic © 2014 Xiang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xiang et al. Molecular Brain 2014, 7:15 Page 2 of 17 http://www.molecularbrain.com/content/7/1/15 mice that show neurodegeneration accelerates Aβ plaque AChE with neurexin clusters (Figure 1A-1, right panels, formation [15]. However, the molecular mechanism or and Figure 1A-2b). Immunoblotting assays (Figure 1B-1) mechanisms by which anomalous AChE contributes to confirmed that BW284c51 treatment increased AChE the pathogenesis of AD remain uncertain. expression but decreased neurexin expression in neurons A progressive loss of synapses in the cortex and hippo- (Figure 1B-2). campus is characteristic of early-stage AD [16]. Synaptic We then conducted reciprocal immunoprecipitation of development and stability are regulated by the interaction neurexin and AChE from lysates of cultured hippocampal of neurexin and neuroligin, transmembrane proteins that neurons. These assays demonstrated that immunopre- are expressed in the pre- and post-synaptic membrane do- cipitation of AChE using an AChE antibody led to co- mains of neurons, respectively [17,18]. These two proteins precipitation of neurexin proteins which exhibited two connect via their ectodomains to form a trans-synaptic bands; and BW284c51 treatment increased this co- junction. Neurexin and neuroligin also interact, via their precipitation (Figure 1C). In turn, a neurexin antibody, cytoplasmic tails, with the PDZ domains of specific scaf- but not IgG protein, co-precipitated a single AChE band folding proteins in the pre- and post-synaptic compart- at about 68 kDa (Figure 1D). These results implied that ments, respectively. In this way, the neuroligin–neurexin AChE molecules are able to interact with some neurexin junctions promote trans-synaptic adhesion and assist in molecules in primary hippocampal neurons. the assembly of pre- and post-synaptic specializations [19]. Interestingly, the molecular sequence and three- Regulation of AChE–neurexin interaction by dimensional structure of the ectodomain of neuroligins protein glycosylation are homologous to the corresponding aspects of AChE Mammalian neurons express both AChE-R and AChE-S, [20]. We previously showed that over-expression of AChE with each isoform displaying distinctive properties in alters the expression of neurexins and neuroligins and molecular assemblies [25]. To study which AChE iso- decreases the number of glutamatergic synapses in hippo- form interacts with neurexin-1β, we expressed Nrxn- campal neurons [21]. The objective of the present study 1β-1’-His with either hAChE-S or hAChE-R in HEK293 was to determine if increased expression of AChE leads cells. We began with outlining the expression profiles to a decrease in the neurexin–neuroligin junctions and of these proteins in the transfected cells. consequently to a reduction in glutamatergic synapses. Consistent with the results of a previous study [26], immunoblotting the lysates of hAChE-S transfected cells, Results using anti-AChE, revealed a dense band at molecular Interaction between AChE and neurexins in weight about 136 kDa (Figure 2A, right lane), as well as Hippocampal neurons two lighter bands at molecular weights about 66 and We first investigated whether AChE interacts physically 68 kDa, respectively (see illustrations in Figure 2A’). with neurexins. Specifically, we performed immunostaining The 66- and 68-kDa bands correspond to monomers of for extracellular AChE in living neurons, followed by coun- AChE-S [27-29], whereas the 136-kDa band may represent terstaining for neurons under membrane-permeabilized dimers of AChE-S. Blotting the lysates of hAChE-R conditions. Consistent with the results of an earlier study transfected cells with anti-AChE also revealed two pro- [22], we found that membrane-bound extracellular AChE tein bands at molecular weights about 68 and 70 kDa molecules assembled in small bunches along neurites (Figure 2A, middle lane; also see Figure 2A’), both of which and were also found in larger patches associated with should be globular monomers, as hAChE-R lacks the the perikaryon (Figure 1A-1, middle panels). In contrast, domain for polymerization. In addition, immunoblotting as previously reported [23], neurexin immunoreactivity assays revealed that the hAChE-S and hAChE-R proteins was diffusely distributed in the perikarya, with small had very similar profiles in the culture medium of trans- clusters in some neurites (Figure 1A-1, left panels). AChE fected HEK293 cells (Figure 2B). Ellman esterase assays immunoreactive particles were co-localized primarily with revealed that under our experimental conditions, the immunofluorescent clusters of neurexin (Figure 1A-1, activity of hAChE in the culture media was about 1.0–1.5 right panels). Treating neurons with the AChE inhibitor units/ml for hAChE-S and 2.0 units/ml for hAChE-R. BW284c51 decreased the fluorescent intensity of dispersed To study the glycosylation pattern of AChE in mam- neurexins in most subcellular compartments but increased malian cells, lysate of HEK293 cells transfected with the fluorescent intensity of neurexin clusters in neurites AChE-R was treated with N-orO-glycohydrolases, both (Figure 1A-1, left panel, inset). Inhibition of AChE activity separately and in combination (Figure 2C). In another increases the expression of AChE [24]. Indeed, treating set of experiments,
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