The goal of this proposal is to understand the molecular components in the formation and maintenance of that neuromuscular synapse. The investigators plan to take advantage of the transgenic mouse technologies to perturb and thereby ascertain the function of three synapse specific genes, the epsilon subunit of the ACh receptor, the 43K/RAPsyn, and the S-laminin.
The specific aims are as follows:
In Specific Aim 1 is to determine the functional consequence of constitutive expressing the gamma (embryonic) or/and inactivation of the epsilon (adult) subunit of the ACh receptor in adult muscle. This will test the hypotheses concerning the biological role and physiological importance of the switch in expression from gamma to epsilon subunits in developing muscle. To this end, transgenic mouse lines expressing high constitutive levels of the ACh receptor gamma subunit, or transgenic mouse lines with the epsilon subunits gene inactivated, will be generated. Overt phenotypes such as weakness, fatigue and behavioral abnormalities which might be associated with a myasthenic syndrome will be assayed. Functional expression of the gamma-subunit will be determined by analyzing the time course of mepc (miniature end plate current) decay; whether, or not gamma-subunit expression is associated with cytopathic abnormalities of the NMJ (neuromuscular junction) will be determined; the possibility that persistence of gamma-subunit expression delays or abolishes the elimination of polyneuronal innervation will be examined.
In Specific Aim 2 is to determine the consequence of disrupting the function of the 43K/RAPsyn gene. 43K/RAPsyn is highly concentrated beneath the synaptic plasma membrane and is thought to interact directly with the ACh receptor to limit mobility. To test this hypothesis, the transgenic mice lines that overexpress mutant 43K/RAPsyn, or transgenic lines with the 43K/RAPsyn gene inactivated, will be generated. These lines will be used to determine whether high density ACh receptor clusters and synaptic localization occur in the absence of RAPsyn; whether the ACh receptor degradation is altered in RAPsyn-less adult muscle; and whether synaptic ultrastructure, including synaptic folds, form normally in the absence of RAPsyn protein.
In Specific Aim 3 is to overexpress or to eliminate the expression of S-laminin in developing and regenerating NMJ's in transgenic mice. S-laminin is a synapse specific component of the basal lamina which may be involved in pre-and post-synaptic membrane interaction. In the transgenic lines overproducing S-laminin, the localization or delocalization of the transgene product S-laminin will be determined. If S-laminin is delocalized, whether there is an effect on the precision of re-innervation will be investigated. Finally, the gross morphological developmental defects caused by S-laminin disruption will be characterized.
| Cho, S I; Ko, J; Patton, B L et al. (1998) Motor neurons and Schwann cells distinguish between synaptic and extrasynaptic isoforms of laminin. J Neurobiol 37:339-58 |
