Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function

Abstract

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.

Figure 1

Generation of a ColQ mutant. (a) The targeted alleles. The top line shows schematic of the ColQ protein, including the amino-terminal PRAD domain that links ColQ to the catalytic subunit of AChE. The second line shows gene structure, with exons indicated by dark boxes and introns by lines. The third and fourth lines show the PRAD1 and PRAD2 vectors, in which the exon encoding the PRAD domain was deleted. The fifth and sixth lines, labeled recombinant, show predicted structures of the mutant alleles. Location of probe used for Southern blots is indicated. (b) Southern blots of wild-type ES cells and homologous recombinant PRAD1 and PRAD2 clones. In the recombinants, the 3.2-kb EcoRI fragment from the wild-type gene is shifted to 4.6 kb. (c) PCR assay used to genotype ColQ mutants. (d) Weight of ColQ ^−/− mice (bottom line) and littermates (top line). Mutant homozygotes are smaller than littermates by P5, and weigh less than half as much as littermates by P20. Bars show mean ± SEM. n = 4–14.

Figure 2

ColQ ^−/− endplates lack AChE. Sections of skeletal muscle from ColQ ^+/− and ColQ ^−/− littermates were double-labeled with rhodamine-α-bungarotoxin plus antibodies to either the catalytic (AChE_T) or collagenous (ColQ) subunit of AChE. Homozygous mutant endplates lacked not only ColQ but also all detectable AChE_T. Bar, 10 μm.

Figure 3

Mepps recorded in muscle fibers from 40-d-old ColQ ^+/− (top) and ColQ ^−/− (bottom) mice. Addition of the AChE inhibitor fasciculin-2 (320 nM) to the bath increased the amplitude and prolonged the decay time constant of mepps in control muscle but had no significant effect on the size or shape of mepps in ColQ ^−/− muscle. Each trace represents the average of 50 mepps. The decay phases of averaged mepps were fitted by a single exponential from 10% to 90% of the maximal amplitude (vertical lines). The difference in mepp amplitudes between ColQ ^+/− and ColQ ^−/− muscles is due to differences in muscle fiber diameter and input resistance.

Figure 4

Molecular forms of cholinesterase in neonatal ColQ mutant muscles. DS and HS pools of cholinesterase, which correspond to globular and asymmetric forms, respectively, were separated on sucrose gradients. Fractions were incubated in the presence of the BuChE inhibitor iso-OMPA (a and b) or the AChE inhibitor BW284c51 (d and e), to selectively assay AChE or BuChE, respectively. a–e show profiles from representative gradients. In each case, ColQ ^−/−, ColQ ^+/−, and ColQ ^+/+ littermates were assayed in parallel, and with identical incubation times. High background levels in e reflect low specific activity, which necessitated overnight incubation of samples. c shows average AChE activities from all experiments, expressed as total activity in International Units (IU) per muscle. In the absence of ColQ, muscles are devoid of asymmetric forms of AChE and BuChE. Levels of G₄ ^na AChE are also dramatically reduced. At P20, asymmetric AChE and BuChE were also completely absent in ColQ ^−/−, but G₄ AChE was present.

Figure 4

Molecular forms of cholinesterase in neonatal ColQ mutant muscles. DS and HS pools of cholinesterase, which correspond to globular and asymmetric forms, respectively, were separated on sucrose gradients. Fractions were incubated in the presence of the BuChE inhibitor iso-OMPA (a and b) or the AChE inhibitor BW284c51 (d and e), to selectively assay AChE or BuChE, respectively. a–e show profiles from representative gradients. In each case, ColQ ^−/−, ColQ ^+/−, and ColQ ^+/+ littermates were assayed in parallel, and with identical incubation times. High background levels in e reflect low specific activity, which necessitated overnight incubation of samples. c shows average AChE activities from all experiments, expressed as total activity in International Units (IU) per muscle. In the absence of ColQ, muscles are devoid of asymmetric forms of AChE and BuChE. Levels of G₄ ^na AChE are also dramatically reduced. At P20, asymmetric AChE and BuChE were also completely absent in ColQ ^−/−, but G₄ AChE was present.

Figure 5

Sections of muscle from P20 ColQ ^−/− and ColQ ^+/− littermates stained for cholinesterase activity by the Karnovsky method. a–h are longitudinal sections; i–r are cross-sections. (a and b) Levels of cholinesterase activity are much higher at control than ColQ ^−/− endplates. (c, d, i, and j) BW284c51 (BW), a selective inhibitor of AChE, has no effect on synaptic cholinesterase activity in ColQ ^−/− muscle, but decreases activity in controls to a level equivalent to that in the mutant. (e and f) Iso-OMPA, a selective inhibitor of BuChE, has only a slight effect on cholinesterase activity in controls but abolishes all activity in mutants. (g and h) No activity is detectable in either muscle in the presence of both BW284c51 and iso-OMPA. These results indicate that both AChE and BuChE are present at control endplates, but only BuChE at ColQ ^−/− endplates. (k and l) Incubation of sections with collagenase to release asymmetric enzyme before staining for BuChE (BW+) reduces activity in controls but has no effect at ColQ ^−/− synapses. (m and n) After incubation with Triton X-100 to release membrane-bound enzyme, some BuChE activity persists at control synapses but is abolished at ColQ ^−/− synapses. (o and p) No BuChE activity is detectable in controls or mutants after sequential treatment with Triton and collagenase. (q and r) 3 d after nerve section, when nerve terminals have degenerated, BuChE persists at control synaptic sites but is lost from mutant synaptic sites. Bar, 20 μm.

Figure 6

Geometry of synapses in ColQ ^−/− mice. Longitudinal sections of P20 control (a) and ColQ ^−/− muscle were stained with rhodamine-α-bungarotoxin. In controls, the postsynaptic membrane contains a set of interconnected AChR-rich gutters. In mutants, some synaptic sites are relatively normal in appearance (b), other appears immature (c), and still others appear fragmented (d). Bar, 10 μm.

Figure 7

Ultrastructure of synapses in ColQ ^−/− mice. Electron micrographs from P20 control (a), P20 ColQ ^−/− (b), and P180 ColQ ^−/− (c and d) muscles. In controls, nerve terminals lie atop a folded postsynaptic membrane and are capped by processes of Schwann cells. In ColQ ^−/− mutants, the subsynaptic cytoplasm appears necrotic at some synapses (b), and some nerve terminals are enwrapped by Schwann cells (d). Even at 6 mo of age, however, some mutant synapses appear normal (c). Bar, 1 μm.

Figure 8

Molecular architecture of synapses in ColQ ^−/− mice. Sections from ColQ ^−/− and control muscle were doubly stained with rhodamine-α-bunagrotoxin (a′–l′) plus antibodies to α-sarcoglycan (a and b), β-dystroglycan (c and d), rapsyn (e and f), utrophin (g and h), or agrin (i and j), or with the lectin VVA-B4 (k and l). No qualitative differences were apparent between mutant and control synapses. Bar, 10 μm.

Figure 9

ColQ is required for assembly of asymmetric forms of AChE in heart and brain. HS extracts of heart (a) or brain (b) of 2-mo-old mice were fractionated on sucrose gradients and assayed for AChE. Only portions of the gradient corresponding to 8–20S material are shown. No asymmetric AChE was detected in ColQ ^−/− tissue.