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  • The AChR is composed of five

    2024-04-22

    The AChR is composed of five homologous, membrane-spanning subunits. AChRs containing two α, one β, one δ and one γ subunit (AChRγ) predominate during embryonic development and mice lacking AChRγ die at birth (Takahashi et al., 2002). After birth, the AChRγs are replaced during the first 2 postnatal weeks by adult-type AChRεs that contain an ε subunit instead of the γ subunit (Mishina et al., 1986, Witzemann et al., 1987, Witzemann et al., 1989). The lack of AChRε cannot be substituted by AChRγ, and mice die prematurely due to severe muscle weakness (Witzemann et al., 1996). Expression of AChRγ in adult muscle is strongly increased in denervated or pharmacologically paralyzed muscle (Witzemann et al., 1991). The signals that trigger the γ-to-ε subunit switch and thus the differential Probenecid of the two receptor subtypes are unknown. Also unknown is how new AChRs are inserted into the postsynaptic membrane to replace receptors from the existing pool. To address these questions, we established a knock-in reporter mouse line: The AChR mice expresses a γ-GFP fusion polypeptide that forms functionally intact GFP-labeled receptors (AChRγ-GFP), which are correctly targeted to the postsynaptic membrane (Gensler et al., 2001). These knock-in mice are born healthy and develop like wild-type mice without any obvious phenotype. However, transcription of the modified γ subunit gene is reduced. Nevertheless, AChRγ-GFP are expressed and can be directly visualized to follow the formation of endplates and nerve/muscle contacts during embryonic development. Therefore, we used the AChR mouse model to determine whether channel conversion is under neuronal control, how AChRγ-GFP are substituted by AChRε at individual endplates and whether this process is synchronized in a muscle or in groups of endplates. Around birth, the number of motor neurons has decreased by over 50% due to programmed cell death, which is thought to be influenced by trophic support provided by muscles as well as neuromuscular activity (Oppenheim, 1991). So far it is, however, not known how individual components of the NMJ affect motor neuron number and axon branching. We examined specifically how reduced receptor densities affect the process of innervation and show that AChRs act as “system-matching” regulators at the synapse.
    Results
    Discussion The AChR mice were generated to study the events that occur during conversion of embryonic-type AChRγ to adult-type AChRε. Its genuine promoter regulates the genetically modified γ subunit gene. Exon 4 was fused to corresponding sequences of a γ-GFP subunit cDNA construct (Gensler et al., 2001). A PGKneo cassette was located downstream to the polyadenylation signal and should therefore not affect the transcriptional efficiency of the γ subunit promoter. The γ subunit gene transcription, however, is reduced in AChR mice, and at this point it is not clear whether this is caused by fusing genomic sequences with cDNA and/or by the presence of the PGKneo cassette. Nevertheless, AChRγ-GFPs are expressed and mark the positioning and differentiation of neuromuscular junctions. Their direct visualization yields new insights in AChRγ-GFP/AChRε dynamics during development and at individual endplates. Reduced AChR densities in developing muscle, however, will decrease the extent of membrane depolarization and embryonic muscle movements, and these factors regulate motor axon branching and motor neuron survival (Oppenheim et al., 2000). The AChR mice represent therefore a new experimental model to study the role of neuromuscular motor endplates, specifically the AChR itself, in regulating innervation. In contrast to previously used paradigms, neuromuscular activity is transiently reduced without employing toxins or inserting deleterious mutations in synaptic regulators that may induce secondary effects or interfere severely with synaptogenesis.
    Experimental methods
    Acknowledgments