Laura E. Paraoanu

Acetylcholinesterase in cell adhesion, neurite growth and network formation

The expression of acetylcholinesterase is not restricted to cholinergically innervated tissues and relates to both neurotransmission and multiple biological aspects, including neural development, stress response and neurodegenerative diseases. Therefore, the classical function of acetylcholinesterase has to be distinguished from its non‐classical, e.g. enzymatic from non‐enzymatic, functions. Here, the roles of acetylcholinesterase in cell adhesion, promoting neurite outgrowth and neural network formation are reviewed briefly, together with potential mechanisms to support these functions. Part of these functions may depend on the structural properties of acetylcholinesterase, for example, protein–protein interactions. Recent findings have revealed that laminin‐1 is an interaction partner for acetylcholinesterase. The binding of acetylcholinesterase to this extracellular matrix component may allow cell‐to‐cell recognition, and also cell signalling via membrane receptors. Studies using monolayer and 3D spheroid retinal cultures, as well as the acetylcholinesterase‐knockout mouse, have been instrumental in elaborating the non‐classical functions of acetylcholinesterase.

Mouse acetylcholinesterase interacts in yeast with the extracellular matrix component laminin-1β

Acetylcholinesterase (AChE) is likely to have roles other than the hydrolysis of acetylcholine, e.g., related to developmental processes like neurite outgrowth, differentiation and adhesion. Here, we investigated whether AChE can function as a heterophilic cell adhesion molecule and searched for proteins interacting with it. Using the yeast two‐hybrid method and a mouse brain cDNA library, we have identified an interaction between a partial cDNA encoding the globular domain IV of laminin chain β1 and the amino acids 240–503 of mouse AChE. Biochemical co‐immunoprecipitation assays confirmed the genetic results. We suggest that AChE, by interacting with laminin‐1, is able to exert changes in adhesion signaling pathways.

On functions of cholinesterases during embryonic development

Expression of cholinesterase (ChE) activity during phases of embryonic development is a general phenomenon in embryonic tissues. To elucidate the role(s) of ChEs during embryonic development, one line of research followed the assumption of a primitive muscarinic system involved in morphogenesis (Hohmann et al., 1995). This means that ChE functioning during development fits into the classical cholinergic neurotransmitter system: acetylcholine (ACh), as a signal, binds to ACh receptors and then is degraded by acetylcholinesterase (AChE) as the terminating enzyme. However, this is just one of the possible mechanisms. The other line of research was driven by evidence for noncholinergic functions of ChE proteins (AChE and butyrylcholinesterase [BChE]). There is accumulating data that other sites on AChE could exert nonclassical roles related to cell differentiation, neurite outgrowth, and adhesion.

Expression patterns of neurexin-1 and neuroligins in brain and retina of the chick embryo: Neuroligin-3 is absent in retina.

Neuroligins (NLs) constitute a family of cell-surface proteins that interact with neurexins (beta-Nxs), another class of neuronal cell-surface proteins, one of each class functioning together in synapse formation. The localization of the various neurexins and neuroligins, however, has not yet been clarified in chicken. Therefore, we studied the expression patterns of neurexin-1 (Nx-1) and neuroligin-1 and -3 during embryonic development of the chick retina and brain by reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). While neurexin-1 increased continuously in both brain and retina, the expression of both neuroligins was more variable. As shown by ISH, Nx-1 is expressed in the inner half retina along with differentiation of ganglion and amacrine cells. Transcripts of NL-1 were detected as early as day 4 and increased with the maturation of the different brain regions. In different brain regions, NL-1 showed a different time regulation. Remarkably, neuroligin-3 was entirely absent in retina. This study indicates that synaptogenetic processes in brain and retina use different molecular machineries, whereby the neuroligins might represent the more distinctly regulated part of the neurexin-neuroligin complexes. Noticeably, NL-3 does not seem to be involved in the making of retinal synapses.

Cell‐by‐cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a ‘cell‐by‐cell’ manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self‐organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R‐spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)‐specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell‐free spaces developed into inner plexiform layer (IPL)‐like areas with extended parallel CR+ fibres. Occasionally, IPL areas merged to combine an ‘inner half retina’, whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE‐spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1–2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT‐PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self‐organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.

Cytochrome-c oxidase is one of several genes elevated in marginal retina of the chick embryo.

The retinal ciliary margin is particularly relevant for the correct generation and regeneration of vertebrate retinae, since pluripotent stem cells are located there throughout development, and--at least in some species--even until adult stages. Our aim was to identify factors (genes) which are involved in processes of proliferation and differentiation in the developing chicken retina. Reverse transcription-polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic retina. Candidate genes analyzed through sequencing and database searches were confirmed by Northern blot analysis and histochemistry. A series of differentially expressed genes were detected, including a neuronal cell adhesion molecule, an esterase, and homeobox gene products. One of the sequenced products was identified as subunit I of cytochrome-c oxidase (COX-1), an enzyme which is central to energy metabolism and particularly relevant for developing nervous systems. Northern blot analysis confirmed its up-regulation in the chick peripheral retina, being maximal at embryonic day 7. In the retinal pigmented epithelium its expression is lower than in the retinal periphery but higher than in central retina. COX histochemistry revealed distinct laminar patterns in central retina, but also an elevated level of activity in the peripheral retina throughout development. These data not only show that the developing ciliary margin of the chick retina has high energy requirements, but also indicate that COX-1 could play essential roles in developing cells and in stem cells of the eye periphery.