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.

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line.

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.

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.

Mouse AChE binds in vivo to domain IV of laminin-1beta.

Functions of acetylcholinesterase (AChE) other than hydrolysis of acetylcholine, e.g. related to cell differentiation, synaptogenesis, neuronal signaling and diseases are well documented, but mechanisms supporting them are not understood. Therefore, we searched for AChE ligands in the central nervous system using a yeast two-hybrid screen (Y2H). 18 independent candidates were identified. One of the membrane or extracellular proteins was laminin-1, an extracellular matrix protein involved in neuronal differentiation and adhesion. The laminin-1 fragment found to interact with AChE contains 898 bp from the 1beta-chain. Moreover, we identified the region containing amino acid residues 240-503 of AChE as essential for interaction with laminin-1beta. Co-immunoprecipitation experiments confirmed the yeast two-hybrid results.

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

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 beta1 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.

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.