A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down‐Regulation Toward Maturity and After Denervation

Abstract

Abstract:In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was~-~1O5 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were~5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation. Key Words: Butyryicholinesterase-Collagen-like tail-Muscle development-Neuromuscular junctions-Postsynaptic specializations.

P2Y2Receptor Activation Regulates the Expression of Acetylcholinesterase and Acetylcholine Receptor Genes at Vertebrate Neuromuscular Junctions

“…The selectivity of the P2Y 1 and the P2Y 2 antibodies in immunoblotting was verified by incubation of each (2 g) with 10 g of its peptide antigen for 1 h at 4°C before applying them to the blots. The following primary antibodies were also used on the same or parallel samples: the anti-mouse AChE antibody (BD Transduction Laboratories, San Diego, CA), at 1:10,000 dilution; the anti-chick AChE antibody (Tsim et al, 1997), at 1:5000; the anti-␣-tubulin antibody (Sigma-Aldrich), at 1:50,000 dilution. The peroxidase-conjugated secondary antibodies used at 1:5000 dilutions in each case were from Zymed Laboratories (South San Francisco, CA).…”

“…Gastrocnemius muscle at different developmental stages from chicken and rat or from adult Xenopus was dissected and rapidly frozen in isopentane/liquid nitrogen. Sections (15 m) were cut, fixed in 2% paraformaldehyde/PBS (15 min at room temperature), and briefly washed with PBS, as detailed by Tsim et al (1997). Astrocytoma cultures were cultured on coverslips until confluence (Sellers et al, 2001) and fixed similarly.…”

“…Where additional blocking by the relevant peptide antigen was to be tested, pretreatment of the antibody with it was as described above. Reaction with each antibody was followed by washes and application of a secondary antibody conjugated to fluorescein-5-isothiocyanate or (on astrocytoma cultures) to cyanine-3, by methods described previously for other antibodies (Tsim et al, 1997;Sellers et al, 2001). The muscle sections were doublelabeled for P2Y 1 receptor and for AChR with 10 nM tetramethylrhodamine-labeled ␣-bungarotoxin (TMR-BuTX; Molecular Probes, Eugene, OR; Tsim et al, 1997).…”

“…Reaction with each antibody was followed by washes and application of a secondary antibody conjugated to fluorescein-5-isothiocyanate or (on astrocytoma cultures) to cyanine-3, by methods described previously for other antibodies (Tsim et al, 1997;Sellers et al, 2001). The muscle sections were doublelabeled for P2Y 1 receptor and for AChR with 10 nM tetramethylrhodamine-labeled ␣-bungarotoxin (TMR-BuTX; Molecular Probes, Eugene, OR; Tsim et al, 1997). Staining of muscle was viewed under a 20 to 40ϫ objective alternately with phase-contrast and fluorescence optics, the latter using excitation at 555 or 488 nm and emission at 580 or 515 nm for TMR or fluorescein-5-isothiocyanate, respectively.…”

Restricted localization of proline‐rich membrane anchor (PRiMA) of globular form acetylcholinesterase at the neuromuscular junctions – contribution and expression from motor neurons

“…Both asymmetric and globular forms of AChE are known to be produced by muscle cells [11,12], and the presynaptic motor nerve terminals synthesize and secrete AChE at NMJs [13,14]. The predominant form of AChE expressed by motor neurons in chick spinal cord is G 4 AChE [15].…”