AChE Publications

The adhesive role of acetylcholinesterase (AChE): Detection of AChE binding proteins in developing rat spinal cord
Bigbee, J.W. and K.V. Sharma
Neurocehmical Research, 29:2043-2050, 2004
Acetylcholinesterase (AChE) is expressed by dorsal root ganglion (DRG) neurons during developmental periods when their central axons are growing into and through the spinal cord. Importantly, our previous studies have shown that AChE induces DRG axonal outgrowth by an adhesive mechanism and thus, have now employed a blot overlay technique to screen for potential AChE binding proteins in the developing spinal cord. Our results show that: (1) AChE binds to proteins with apparent molecular weights of 200, 110, 35 and 33 kDa; (2) these proteins are developmentally expressed during periods of axonal outgrowth from DRG neurons; (3) all four proteins are synthesized by astrocytes; and (4) AChE binding to these proteins is highly dependent on ionic strength supporting an electrostatic mechanism of adhesion. Taken together, these data provide further documentation for the participation of AChE in adhesive interactions during morphogenesis of the central nervous system and suggest a role for astrocytes in regulating AChE-mediated axonal growth.

Mechanisms underlying the axonal growth promoting property of acetylcholinesterase
Bigbee, J.W., K.V. Sharma, C. Koenigsberger and S. Brimijoin
Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects, Inestrosa, NC and EO Campos (eds.), Diseno e Impresiones J&J Ltd., Chile, pgs. 295-300, 2004.
Acetylcholinesterase (AChE) is transiently expressed in a variety of developing systems during stages when cells are engaged in growth and migration (1). This observation has led to the hypothesis that embryonic AChE plays a morphogenic role during development. While AChE expression has been described during hematopoesis, osteogenesis, and limb and somite formation, this embryonic expression of AChE has been most extensively studied in nervous tissues where its appearance coincides with periods of neuronal differentiation and axonal outgrowth (2,3). Experimental studies have now clearly demonstrated a direct link between AChE and axonal growth, however, the mechanisms underlying this morphogenic ability are not fully understood. Our laboratory is examining both the protein-protein interactions and signaling properties of AChE in an effort to elucidate the mechanisms underlying its growth promoting ability.

Direct evidence for an adhesive function in the non-cholinergic role of acetylcholinesterase in neurite outgrowth.
Sharma, KV, C. Koenigsberger, S. Brimijoin and J.W. Bigbee
Journal of Neuroscience Research,63:165-175, 2001.
Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell-surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChET-cDNA in either the sense or antisense orientations to overexpress or knockdown AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that over-expression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons
Bigbee, J.W., K.V. Sharma, E.L-P. Chan, O. Bogler
Brain Research, 861:354-362, 2000.
Acetylcholinesterase (AChE) can promote neurite outgrowth through a mechanism that is independent of its role in hydrolyzing the neurotransmitter acetylcholine. It has been proposed that this neuritogenic capacity of AChE may result from its intrinsic capacity to function in adhesion. In this report we directly tested this hypothesis using neuroblastoma cell lines which have been engineered for altered cell-surface expression of AChE. Using a microtiter-plate adhesion assay and the Electrical Cell-Substrate Impedance Sensing (ECIS) method we demonstrate that the level of cell-substratum adhesion of these cells directly correlates with their level of AChE expression. Furthermore, this adhesion is blocked by either an anti-AChE antibody or a highly specific AChE-inhibitor (BW284c51), both of which have also been shown to block neurite outgrowth. In addition, cells which over-express AChE showed enhanced neurite initiation. By employing cell lines with different levels of AChE expression in two types of cell-substratum adhesion assays, our current studies provide evidence for an adhesive function for AChE. These results, together with the fact that AChE shares sequence homology and structural similarities with several known cell adhesion molecules, support the hypothesis that AChE may promote neurite outgrowth, at least in part, through an adhesive function.

Morphogenic role for AChE in axonal outgrowth during neural development.
Bigbee, J.W., K.V. Sharma, J.J. Gupta and J.L. Dupree.
Environmental Health Perspectives, 107(Suppl 1) 81-87, 1999.
Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions. However, results from our laboratory and others indicate that AChE has an extra-synaptic, non-cholinergic role during neural development. In this report we review our findings which demonstrate a morphogenic role for AChE using a neuronal cell culture model and discuss how these data suggest that AChE has a cell adhesive function during neural development. These results could have additional significance since AChE is the target enzyme of agricultural organophosphate and carbamate pesticides as well as the commonly used household organophosphate, chlorpyrifos (Dursban). Prenatal exposure to these agents could potentially have adverse effects on neural development by interfering with the morphogenic function of AChE.

A morphogenic role for acetylcholinesterase in neurite outgrowth: Studies on a cell adhesive function
Bigbee, J.W. and K.V Sharma.
In Structure and Function of Cholinesterases and Related Proteins, Doctor, B.P., D.M. Quinn, R.L. Rotundo and P. Taylor, (eds.), Plenum Press, pgs. 577-584, 1998.
During the past ten years there has been increasing interest in non-cholinergic roles for acetylcholinesterase (AChE) during neural development, specifically its role in axonal outgrowth. Building on descriptive data in developing chick, rat and primate nervous systems, our laboratory and others are providing a more direct link between AChE expression and process outgrowth through the use of in vitro systems. We have previously shown that non-cholinergic, rat dorsal root ganglion (DRG) neurons, express AChE and that the level of AChE increases in parallel with neurite outgrowth in culture. We have also shown that treatment with certain AChE inhibitors produces a dose-dependent, but reversible, reduction in outgrowth which is accompanied by altered neurite morphology and abnormal accumulations of neurofilaments in the cell body. Similar effects on outgrowth have also been reported for other PNS and CNS neurons. Overexpression of AChE has been shown to induce process growth and conversely, treatment with AChE antisense oligodeoxynucleotides results in reduced neuritic outgrowth. While a mechanism underlying the morphogenic function of AChE has yet to be defined, these data are consistent with a cell adhesive role for AChE. This hypothesis, originally proposed by Paul Layer and colleagues is supported by data showing that AChE shares amino acid sequence homology with known cell adhesion molecules. In this regard, we have previously shown that the level of AChE expression is higher when DRG neurons are grown on substrata which are less permissive to outgrowth, suggestive of a role in cell-substratum interactions. In this report we review our studies demonstrating the effects of AChE monoclonal antibody treatment on both neurite outgrowth and attachment in primary cultures of DRG neurons. We also examine the effects on neuronal attachment when the culture substratum is supplemented with exogenous AChE. Finally, we propose potential adhesive mechanisms by which AChE may function in a morphogenic role.

Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: further evidence for a cell adhesive role for neuronal acetylcholinesterase
Sharma K.V. and J.W. Bigbee
Journal of Neuroscienc Research, 53:454-64, 1998.
Data from our laboratory and others demonstrate that acetylcholinesterase(AChE) is expressed transiently by neurons during periods of neurite outgrowth preceding synaptogenesis, suggesting an extrasynaptic function for this molecule. These findings, along with reports that AChE shares amino acid sequence homology and structural similarities with known cell adhesion molecules, have led to the theory that, during development, AChE may exert a morphogenic effect through cell adhesion. To further test this hypothesis, we have examined the effects of an AChE monoclonal antibody (MAB304) on neurite outgrowth in primary cultures of rat dorsal root ganglion (DRG) neurons. Short-term, high-concentration antibody treatment produced a rapid detachment of established DRG neurites, which was followed by regrowth upon removal of the antibody from the culture medium. This effect appeared to be site-specific, because other AChE antibodies that were able to detect AChE immunocytochemically failed to produce this disadhesion. Long-term, low-concentration antibody exposure produced a 50% reduction in total area of outgrowth, in which neurites were more densely packed and interlaced compared with the neurites in control cultures. These results extend our previous observations on the outgrowth perturbing effects of AChE inhibitor treatment and provide further evidence that AChE
may support neurite outgrowth through a cell adhesive role.

Acetylcholinesterase inhibitor treatment delays recovery from axotomy in cultured dorsal root ganglion neurons
Dupree J.L. and J.W. Bigbee
Journal of Neurocytology, 25:439-54, 1996.
We have previously reported that dorsal root ganglion neurons cultured in the presence of the highly specific, reversible acetylcholinesterase inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide(BW284c51), showed significantly reduced neurite outgrowth and contained massive perikaryal inclusions of neurofilaments. In the present report we have more closely examined these changes in a time course study over a 21-day culture period using a combined morphological, immunocytochemical and enzymatic approach and additionally, describe, the effects of acetylcholinesterase inhibitor treatment on the state of neurofilament phosphorylation. Finally, we have examined the effects of co-administration of N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) with BW284c51. At 1 day in culture, both control and treated cells displayed eccentrically located nuclei, numerous polysomes and perikaryal accumulations of neurofilaments which were immunoreactive with both phosphorylation- and nonphosphorylation-dependent neurofilament antibodies. These cytological changes, which are common features of the chromatolytic reaction following axotomy in vivo, rapidly resolved in the control neurons, where by 7 days in culture, the neurofilament accumulations had completely disappeared and neurite outgrowth was robust. In contrast, inhibitor-treated neurons retained the post-axotomy features up to 21 days and had significantly reduced neurite outgrowth. In addition, we have investigated a possible role of cyclic adenosine monophosphate (cAMP) in the recovery process since it has been shown to enhance neuritic outgrowth in cultured neurons. Our results demonstrate that the addition of dbcAMP, a membrane permeable analog of cAMP, significantly enhanced neuritic outgrowth and accelerated the recovery of BW284c51-treated dorsal root ganglion cells, as gauged by the disappearance of the axotomy-related cytological changes. Treatment with dbcAMP also increased acetylcholinesterase activity which has been positively correlated with neurite outgrowth both in vivo and in vitro. Together, these observations suggest that acetylcholinesterase has a non-cholinolytic, neurotrophic role in neuronal regeneration and development.

Inverse correlation of acetylcholinesterase (AChE) activity with the presence of neurofilament inclusions in dorsal root ganglion neurons cultured in the presence of a reversible inhibitor of AChE
Dupree J.L., E.N. Maynor and J.W. Bigbee
Neuroscience Letters, 197:37-40, 1995.
We have previously shown that treatment of cultured dorsal root ganglion neurons (DRGN) with a highly specific, reversible acetylcholinesterase (AChE) inhibitor, BW284c51, retards neuritic outgrowth in a dose dependent manner and is accompanied by the presence of abnormal, perikaryal neurofilament (NF) inclusions in approximately 40% of the cells. Since subpopulations of DRGN have been classified according to their levels of AChE activity, we have combined immunocytochemical and enzyme histochemical techniques to investigate a possible correlation between AChE activity and the presence of NF inclusion formation. Our results show that after inhibitor treatment, cells with low levels of AChE activity have a greater percentage of inclusions, with nearly 75% of cells with undetectable levels of AChE activity containing inclusions. In contrast, inclusions were present in only 3.2% of cells with high levels of AChE activity. This inverse relationship between AChE activity and the presence of NF inclusions supports our previous observations that this enzyme may have extra-synaptic functions which could affect neuronal development and regeneration.

Retardation of neuritic outgrowth and cytoskeletal changes accompany acetylcholinesterase inhibitor treatment in cultured rat dorsal root ganglion neurons
Dupree J.L. and J.W. Bigbee JW.
Journal of Neuroscience Research, 39:567-75, 1994.
Over the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non-cholinergic and non-cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non-cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum Matrigel and the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE-specific inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) ranging from 10(-4) to 10(-7) M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on Matrigel at 10(-5) and 10(-4) M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10(-6), 10(-5), and 10(-4) M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51-treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseases such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body.

Substratum-induced modulation of acetylcholinesterase activity in cultured dorsal root ganglion neurons
Gupta J.J. and J.W. Bigbee
Journal of Neuroscience Research, 31:454-61, 1992.
Acetylcholinesterase (AChE) has been shown to be transiently expressed in the developing nervous system during periods of neuronal migration and axonal outgrowth. We are investigating the possible interaction of substratum with AChE activity in dorsal root ganglion neurons (DRGN) cultured on substrata with varying degrees of permissiveness for neurite outgrowth: (1) extracellular matrix substrata: reconstituted basal lamina Matrigel (MGEL), laminin (LAM) and type I collagen (COL), and (2) organotypic substrata: unfixed, frozen sections of sciatic nerve (SN) and spinal cord (SC). In group 1, histochemical staining for AChE in DRGN was lowest on MGEL where outgrowth was most vigorous, intermediate on LAM, and highest on COL where neurite outgrowth was reduced by 55% compared to Matrigel and highly fasciculated. A similar trend was seen when the cultures were assayed biochemically, 2.84 +/- 0.14 nmoles ACh hydrolyzed/ganglion/hr (MGEL), 4.42 +/- 0.19 (LAM), 5.79 +/- 0.37 (COL). In group 2, SN supported an expansive outgrowth with lower AChE activity than in DRGN grown on SC where outgrowth was minimal. These studies show that the levels of AChE activity can be modulated by substratum, perhaps in proportion to the permissiveness of the substratum to neuritic outgrowth. These results are discussed in relation to possible non-cholinergic roles of AChE.