Author Report for: De Jaco A

The genetics underlying autism spectrum disorder (ASD) is complex and heterogeneous, and de novo variants are found in genes converging in functional biological processes. Neuronal communication, including trans-synaptic signaling involving two families of cell-adhesion proteins, the presynaptic neurexins and the postsynaptic neuroligins, is one of the most recurrently affected pathways in ASD. Given the role of these proteins in determining synaptic function, abnormal synaptic plasticity and failure to establish proper synaptic contacts might represent mechanisms underlying risk of ASD. More than 30 mutations have been found in the neuroligin genes. Most of the resulting residue substitutions map in the extracellular, cholinesterase-like domain of the protein, and impair protein folding and trafficking. Conversely, the stalk and intracellular domains are less affected. Accordingly, several genetic animal models of ASD have been generated, showing behavioral and synaptic alterations. The aim of this review is to discuss the current knowledge on ASD-linked mutations in the neuroligin proteins and their effect on synaptic function, in various brain areas and circuits.

Autism spectrum disorders (ASDs) comprise a heterogeneous group of disorders with a complex genetic etiology. Current theories on the pathogenesis of ASDs suggest that they might arise from an aberrant synaptic transmission affecting specific brain circuits and synapses. The striatum, which is part of the basal ganglia circuit, is one of the brain regions involved in ASDs. Mouse models of ASDs have provided evidence for an imbalance between excitatory and inhibitory neurotransmission. Here, we investigated the expression of long-term synaptic plasticity at corticostriatal glutamatergic synapses in the dorsal striatum of the R451C-NL3 phenotypic mouse model of autism. This mouse model carries the human R451C mutation in the neuroligin 3 (NL3) gene that has been associated with highly penetrant autism in a Swedish family. The R451C-NL3 mouse has been shown to exhibit autistic-like behaviors and alterations of synaptic transmission in different brain areas. However, excitatory glutamatergic transmission and its long-term plasticity have not been investigated in the dorsal striatum so far. Our results indicate that the expression of long-term synaptic depression (LTD) at corticostriatal glutamatergic synapses in the dorsal striatum is impaired by the R451C-NL3 mutation. A partial rescue of LTD was obtained by exogenous activation of cannabinoid CB1 receptors or enhancement of the endocannabinoid tone, suggesting that an altered cannabinoid drive might underlie the deficit of synaptic plasticity in the dorsal striatum of R451C-NL3 mice.

An increasing number of rare mutations linked to autism spectrum disorders have been reported in genes encoding for proteins involved in synapse formation and maintenance, such as the post-synaptic cell adhesion proteins neuroligins. Most of the autism-linked mutations in the neuroligin genes map on the extracellular protein domain. The autism-linked substitution R451C in Neuroligin3 (NLGN3) induces a local misfolding of the extracellular domain, causing defective trafficking and retention of the mutant protein in the endoplasmic reticulum (ER). The activation of the unfolded protein response (UPR), due to misfolded proteins accumulating in the ER, has been implicated in pathological and physiological conditions of the nervous system. It was previously shown that the over-expression of R451C NLGN3 in a cellular system leads to the activation of the UPR. Here, we have investigated whether this protective cellular response is detectable in the knock-in mouse model of autism endogenously expressing R451C NLGN3. Our data showed up-regulation of UPR markers uniquely in the cerebellum of the R451C mice compared to WT littermates, at both embryonic and adult stages, but not in other brain regions. Miniature excitatory currents in the Purkinje cells of the R451C mice showed higher frequency than in the WT, which was rescued inhibiting the PERK branch of UPR. Taken together, our data indicate that the R451C mutation in neuroligin3 elicits UPR in vivo, which appears to trigger alterations of synaptic function in the cerebellum of a mouse model expressing the R451C autism-linked mutation.

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.

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 alpha/beta-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.

The alpha/beta-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the alpha/beta-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the alpha/beta-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler alpha/beta-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the alpha/beta-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the alpha/beta-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. DATABASE: A listing and description of proteins in the alpha/beta-hydrolase fold family of proteins is available at http:\/\/bioweb.supagro.inra.fr/ESTHER/general?what=index.

The alpha/beta 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 alpha/beta 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 alpha/beta-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 alpha/beta-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 alpha/beta-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 alpha/beta-hydrolase fold.

The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.

Proteins of the alpha/beta-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the alpha/beta-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the alpha/beta-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys does not interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization.

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

Mammalian acetylcholinesterase (AChE) gene expression is exquisitely regulated in target tissues and cells during differentiation. An intron located between the first and second exons governs a approximately 100-fold increase in AChE expression during myoblast to myotube differentiation in C2C12 cells. Regulation is confined to 255 bp of evolutionarily conserved sequence containing functional transcription factor consensus motifs that indirectly interact with the endogenous promoter. To examine control in vivo, this region was deleted by homologous recombination. The knock-out mouse is virtually devoid of AChE activity and its encoding mRNA in skeletal muscle, yet activities in brain and spinal cord innervating skeletal muscle are unaltered. The transcription factors MyoD and myocyte enhancer factor-2 appear to be responsible for muscle regulation. Selective control of AChE expression by this region is also found in hematopoietic lineages. Expression patterns in muscle and CNS neurons establish that virtually all AChE activity at the mammalian neuromuscular junction arises from skeletal muscle rather than from biosynthesis in the motoneuron cell body and axoplasmic transport.

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 alpha/beta-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.

A mutation linked to autistic spectrum disorders encodes an Arg to Cys replacement in the C-terminal portion of the extracellular domain of neuroligin-3. The solvent-exposed Cys causes virtually complete retention of the protein in the endoplasmic reticulum when the protein is expressed in transfected cells. An identical Cys substitution was reported for butyrylcholinesterase through genotyping patients with post-succinylcholine apnea. Neuroligin, butyrylcholinesterase, and acetylcholinesterase are members of the alpha,beta-hydrolase fold family of proteins sharing sequence similarity and common tertiary structures. Although these proteins have distinct oligomeric assemblies and cellular dispositions, homologous Arg residues in neuroligin-3 (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcholinesterase (Arg-395) are conserved in all studied mammalian species. To examine whether an homologous Arg to Cys mutation affects related proteins similarly despite their differing capacities to oligomerize, we inserted homologous mutations in the acetylcholinesterase and butyrylcholinesterase cDNAs. Using confocal fluorescence microscopy and analysis of oligosaccharide processing, we find that the homologous Arg to Cys mutation also results in endoplasmic reticulum retention of the two cholinesterases. Small quantities of mutated acetylcholinesterase exported from the cell retain activity but show a greater K(m), a much smaller k(cat), and altered substrate inhibition. The nascent proteins associate with chaperones during processing, but the mutation presumably restricts processing through the endoplasmic reticulum and Golgi apparatus, because of local protein misfolding and inability to oligomerize. The mutation may alter the capacity of these proteins to dissociate from their chaperone prior to oligomerization and processing for export.

An Arg to Cys mutation in the extracellular domain of neuroligin-3 (NL3) was recently found in a twin set with autism [S. Jamain, H. Quach, C. Betancur, M. Rastam, C. Colineaux, I.C. Gillberg, H. Soderstrom, B. Giros, M. Leboyer, C. Gillberg, T. Bourgeron, Paris Autism Research International Sibpair Study, mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism, Nat. Genet. 34 (2003) 27-29]. The Cys substitution in NL3 causes altered intracellular protein trafficking, intracellular retention and diminished association with its cognate partner, beta-neurexin [D. Comoletti, A. De Jaco, L.L. Jennings, R.E. Flynn, G. Gaietta, I. Tsigelny, M.H. Ellisman, P. Taylor, The R451C-neuroligin-3 mutation associated with autism reveals a defect in protein processing, J. Neurosci. 24 (2004) 4889-4893]. NL3, butyrylcholinesterase (BuChE), and acetylcholinesterase (AChE), as members of the (/(-hydrolase fold family of proteins, share over 30% of amino acid identity in their extracellular domains. In particular, Arg451 in NL3 is conserved in the alpha/beta-hydrolase fold family being homologous to Arg386 in BuChE and Arg395 in AChE. A Cys substitution at the homologous Arg in the BuChE was found studying post-succinylcholine apnea in an Australian population [T. Yen, B.N. Nightingale, J.C. Burns, D.R. Sullivan, P.M. Stewart, Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population, Clin. Chem. 49 (2003) 1297-308]. We have made the homologous mutation in the mouse AChE and BuChE genes and showed that the Arg to Cys mutations resulted in identical alterations in the cellular phenotype for the various members of the alpha/beta-hydrolase fold family proteins.

During myogenesis, marked increases in both acetylcholinesterase (AChE) and its encoding mRNA are observed. The intron in the AChE gene between non-coding exon 1 [T.L. Rachinsky, S. Camp, Y. Li, T.J. Ekstrom, M. Newton, P. Taylor, Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species, Neuron 5 (1990) 317-327] and start-site containing exon 2 [A. Mutero, S. Camp, P. Taylor, Promoter elements of the mouse acetylcholinesterase gene, J. Biol. Chem. 270 (4) (1995) 1866-1872] appears to be responsible for the enhanced expression of the enzyme upon myoblast to myotube differentiation. Deletion of a 255 bp sequence within the first intron of the AChE gene abolishes the increase in cell-associated activity observed with differentiation. To study the involvement of the intronic region in post-transcriptional processing of AChE message, we used real time RT-PCR to quantify spliced and unspliced message levels in myoblasts and myotubes. We observe a 200-fold increase of the fully spliced mRNA associated with myotube formation, while the increase in the unspliced mRNA retaining either intron 1 or intron 2 is only 5 to 15-fold. We have generated knockout mice without the conserved region of intron 1. The mice show a phenotype where skeletal muscle, hematopoietic and central nervous system AChE expression differ with the greatest effect existing in the disappearance of skeletal muscle expression [S. Camp, L Zhang, M. Marquez, B. de La Torre, J.M. Long, G. Bucht, P. Taylor, Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion, VIII IMC Proceedings].

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the beta-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished beta-neurexin-1 (NX1beta) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1beta. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.

The possible role of acetylcholine as a modulator of neuronal differentiation has been tested using a neuroblastoma cell line (N18TG2), which does not synthesize any neurotransmitter. Acetylcholine synthesis has been activated in this line by transfection with a construct containing a choline acetyltransferase (ChAT) cDNA; ChAT-positive clones share a higher ability to grow fibers and an activation of synapsin I expression compared to the parental cells. Atropine, a muscarinic antagonist, abolishes the higher ability to grow fibers of ChAT-positive transfected clones, and the cholinergic agonist carbachol induces higher neurite outgrowth in the parental line. In transient transfections of ChAT-positive clones, the expression of a reporter gene under the control of synapsin I promoter is considerably reduced by atropine, while it is not modified by carbachol; in contrast, in the parental cells, which do not synthesize acetylcholine, the reporter gene expression is induced by carbachol and this effect is abolished by atropine. The data presented provide evidence for the existence of a direct modulation of fiber outgrowth and synapsin I expression by muscarinic receptor activation, which may be related to early growth response gene-1 (EGR-1) levels.

The mouse acetylcholinesterase AChE(H) was expressed in the yeast Kluyveromyces lactis. The AChE(H) activity was detectable in intact cells whereas it was absent in the culture media. Glucanase treatment and immunoelectron microscopy data indicated that AChE(H) is anchored to plasma membrane and that the mouse GPI-signaling is compatible with the K. lactis targeting machinery. The AChE(H) was also expressed in a K. lactis strain carrying an inactivated allele of KlPMR1, the gene coding for a P-type Ca(2+)-ATPase of the Golgi apparatus. This mutant displays changes in protein glycosylation and cell wall structure. The AChE(H) activity detected in Klpmr1Delta cells was more than twofold higher than that observed in wild-type cells. The combination of AChE expression and anchoring with the characteristics of Klpmr1Delta strain of K. lactis resulted in yeast cells displaying high AChE activity. This could be regarded as a novel sensing unit to be employed for detecting AChE inhibitors as pesticides.

Antennapedia homeobox peptide has been reported to enhance neurite outgrowth and branching. Thus it is of interest to investigate whether antennapedia peptide is capable of modulating the expression of genes related to different events of neuronal development. In this paper we report the enhancement of a 68 KDa neurofilament subunit, choline acetyltransferase and acetylcholinesterase expression in spinal cord neurons, elicited by antennapedia peptide. Modulation of gene expression is different with respect to each gene product analyzed, suggesting a specific action of the peptide on diverse genes controlling different events of neuronal differentiation.

Development of the nervous system is dependent on the co-operation between cell determination events and the action of epigenetic factors; in addition to well known factors, e.g. growth factors, neurotransmitters have been assigned a role as "morphogens" and modulators of neuronal differentiation in an early developmental phase. The possible role of acetylcholine as a modulator of neuronal differentiation has been considered in two experimental systems. A neuroblastoma cell line, which does not synthesise any neurotransmitter, has been transfected with a choline acetyltransferase construct; activation of acetylcholine synthesis, thus achieved, is followed by a higher expression of neuronal specific traits. The presence in these cells of muscarinic receptors is consistent with the existence of an autocrine loop, which may be responsible for the more advanced differentiation state observed in the transfected cells. Expression of cholinergic markers appears as a common feature of DRG sensory neurons, independently of the neurotransmitter used. Choline acetyltransferase can be detected in DRG at early developmental stages. The distribution of muscarinic receptors in DRG has suggested that activation of acetylcholine synthesis may be related in an early developmental phase to the interaction between neurons and nonneuronal cells and to modulation of cell differentiation. Both systems suggest that acetylcholine may have a role as a modulator of neuronal differentiation.

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.

N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma x glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.