Andrea Robitzki

Of layers and spheres: the reaggregate approach in tissue engineering

The reaggregate approach involves the regeneration of histotypical three-dimensional spheres from dispersed cells of a given tissue in suspension culture. Reaggregated spheres are used as tumour, genetic, toxicological, biohybrid and neurosphere models, and often replace animal experimentation. A particularly instructive example is the use of reaggregation to regenerate complete laminar tissue from avian embryonic retina. By revealing constraints of layered tissue formation, such retinal spheres could be instrumental for regenerative medicine, including stem cell-based tissue engineering.

Butyrylcholinesterase Antisense Transfection Increases Apoptosis in Differentiating Retinal Reaggregates of the Chick Embryo

Abstract: To investigate the roles of the enzymes butyryl‐ and acetylcholinesterase (BChE and AChE) in retinal proliferation and differentiation, we use reaggregated spheres from retinal cells of the 6‐day‐old chick embryo, forming cellular and fibrous areas homologous to all layers of a normal retina. Recently, we could suppress BChE expression by transfecting these so‐called retinospheroids during their proliferation period with a pSVK3 expression vector containing a 5′ fragment of the rabbit BChE gene in antisense orientation. Along with morphological changes, proliferation was significantly decreased. Here, we have studied the effect of antisense BChE suppression during the differentiation period of retinospheroids. As BChE is suppressed, the differentiation of AChE‐positive cells is increased, whereas the immunoreactivities for red and green cone‐specific opsins are strongly reduced. Concomitantly, the rate of apoptosis as determined by propidium iodide uptake, by increased CPP 32‐like caspase expression, and by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling and DNA fragmentation assays is roughly doubled, predominantly at the expense of degenerating photoreceptor precursors. This is further strong evidence that the proliferation marker BChE regulates an intricate balance between cell proliferation, cell differentiation, and programmed cell death in this in vitro retinal system.

BUTYRYLCHOLINESTERASE IS COMPLEXED WITH TRANSFERRIN IN CHICKEN SERUM

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.

Aryl acylamidase activity exhibited by butyrylcholinesterase is higher in chick than in horse, but much lower than in fetal calf serum

Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than horse and 80-fold higher than chick serum. We then show that an immunoisolated preparation of BChE from chicken serum presents significant activity for AAA. Both in sera and with the purified enzyme, the AAA activity is fully inhibited by anticholinesterase drugs, showing that AAA activity is exclusively conveyed by the BChE molecule. Noticeably, AAA inhibition even occurs at lower drug concentrations than that of BChE activity itself. Moreover, AAA is sensitive to serotonin. These data indicate that (1) AAA is a general feature of serum BChE of vertebrates including avians, (2) AAA is effectively inhibited by cholinergic and serotonergic agents, and (3) AAA may have a developmental role, since it is much pronounced in a serum from fetal animals. Functionally, deamination of neuropeptides, a link between cholinergic and serotonergic neurotransmitter systems, and roles in lipoprotein metabolism could be relevant.

Anticholinesterase treatment of chicken retinal cells increases acetylcholinesterase protein independently of protein kinase C

It has been reported that anticholinesterase exposure, e.g. by environmental toxins or nerve gases, can increase acetylcholinesterase (AChE) protein, possibly as an autoregulatory stress response. We earlier have transfected retinal cells of the chick embryo with a pSVK3-AChE(rab)-cDNA vector to heterologously express rabbit AChE, which concomitantly also increased AChE protein from chick. To analyse further the cell-internal pathways of these different paradigms (anticholinesterase treatment vs. AChE transfection) which both lead to an AChE increase, we here show that AChE overexpression by transfection leads to an increase in protein kinase C (PKC). Most remarkably, when cells independently of, or in addition to their transfection are treated with 10 microM of the AChE inhibitor BW284c51, AChE protein levels are much more dramatically increased up to 20-fold. This treatment, however, does not affect PKC. These data show that (i) retinal cells respond to anticholinesterase insult by a massive increase of AChE protein; (ii) the response to BW284c51 is not PKC-mediated; and (iii) both strategies of AChE increase follow different cell-internal pathways, their effects being additive. The ecological and biomedical implications of these findings are briefly discussed.

Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells.

Abstract. Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2xa0days i.c. with a sense pSVK3-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5-bromo-2-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.

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.

Nonenzymatic Roles of Cholinesterases in Avian Neurogenesis

Understanding the functioning of cholinesterases has wide environmental, military and clinical relevance. The already longstanding hypothesis of nonenzymatic roles, which is now gaining momentum, would - once accepted - entirely change our views on all these topics. It is now that this hypothesis receives more attention due to major achievements in at least three diverse fields of cholinesterase research: a) the further elaboration of the three-dimensional structure of cholinesterases and related proteins, b) the finding of homologies between cholinesterases and cell adhesion molecules (1,2), and c) the accumulating experimental evidence for such functions (3,4; this volume). Table 1 presents a selection of some of the experimental studies supporting a nonenzymatic function of cholinesterases in neuritic growth. Four major types of approaches have been taken: perturbation of neurite growth by pharmacological, AChE antibody, ChE antisense intervention, and AChE-overexpression.

Suppression of Butyrylcholinesterase Induces Overexpression of Acetylcholinesterase and Increased Apoptosis in Embryonic Chick Retinospheroids

We here report the generation of a reaggregation system of embryonic chick retinal cells by transfection with sense- and antisense-cholinesterase expression vectors. Reaggregation of embryonic retina cells leads to so-called retinospheroids, which show a high degree of structural development. The cholinesterases butyrylcholinesterase (BChE) as a proliferation marker and acetylcholinesterase (AChE) as a differentiation marker are expressed in embryonic retina in a mutually exclusive manner and seem to be coregulated (1). BChE is expressed before and during mitosis, whereas AChE is expressed about 10 to 15 hours after the last mitosis. During the proliferation period, inhibition of BChE transcription by an antisense strategy suggests that decrease of BChE mRNA promotes an increase of acetylcholinesterase. Moreover, a strong decrease of proliferation and growth of antisense 5′-BChE transfected retinospheroids was apparent (2). These facts demonstrate that AChE is expressed early in neuronal development, however, it is not localized to the neuronal cell membrane until later during the process of synaptogenesis. As detected by vital propidium iodide uptake, TdT-mediated dUTP nick end labeling (TUNEL) and DNA fragmentation assays, the suppression of BChE during the period of differentiation, resulted in an increase of apoptosis. Furthermore, the number of AChE-positive cells increased dramatically, whereas rod- and cone-specific photoreceptor precursor cells decreased. Thereby, red and green cones seem to be completely degenerated (3). These resuits suggest that the proliferation marker BChE determines an intracellular balance between proliferation, differentiation and apoptosis during retinogenesis. The inhibition of BChE induces a higher degree of differentiation of AChE-positive cells, and a higher rate of apoptotic photoreceptor precursors.

Cholinesterases in Neurogenesis

Developmental biology is fundamental to all biomedical research. The credo of developmental biologists is to study the simple state in order to understand the complex, the healthy to understand the diseased, and to follow growth in order to understand the final decay of biological systems. This applies particularly to the study of the most complex organ that has evolved, the human brain and its slow deterioration as observed in dementia. Following this principle, general studies on neurogenesis are a prerequisite for detecting defects in Alzheimer’s disease.