Several lines of evidence suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may promote neuromuscular protection and repair. Thus, proper targeting of CNTF receptor signaling may selectively counteract the devastating effects of neuromuscular disorders by appropriately enhancing these naturally evolved mechanisms. Unfortunately, current understanding of endogenous CNTF receptor function in the adult is extremely limited because blockage of the receptor in vivo (through disruption of the critical CNTF receptor a [CNTFRa] gene) leads to perinatal death. To overcome this problem, we will use Cre/lox techniques to selectively disrupt the CNTFRa gene.
Specific Aim 1 will examine the contribution of CNTF receptor signaling to the survival, protection and phenotype maintenance of adult motor neurons. The CNTFRa gene will be disrupted in facial motor neurons of """"""""floxed"""""""" CNTFRa mice with: 1) stereotaxic injection of the facial nucleus with an adeno-associated virus that directs Cre recombinase (Cre) expression and 2) a gene construct enabling temporally controlled induction of Cre activity. Preliminary data indicate an essential in vivo role for CNTFRa in adult motor neuron survival. We will quantitatively characterize this function with reporter genes, immunohistochemistry and stereological cell counting and also determine whether insult is required to activate this neuroprotective system (as preliminary data suggest), and if so, what forms of insult. In addition, a reporter gene and immunohistochemistry will be used to determine whether CNTF receptor signaling maintains motor neuron cholinergic phenotype in vivo. Finally, signaling proteins potentially involved in the neuroprotection will be identified through in situ hybridization and immunohistochemistry.
Specific Aim 2 will define the role of skeletal muscle CNTFRa in neuromuscular protection and repair. Preliminary data from floxed CNTFRa mice with skeletal muscle specific Cre expression reveal that muscle CNTFRa is required for normal motor recovery following peripheral nerve lesion. We will characterize this functional deficit with """"""""footprint"""""""" analysis and identify the underlying cellular mechanisms by quantifying: 1) neuromuscular junction formation, 2) motor neuron survival and cholinergic phenotype, and 3) muscle contractility. In addition, STAT3-based immunohistochemistry will be used to localize cellular sites of muscle-CNTFRa-dependent signaling after nerve lesion. Moreover, microarray analysis of muscle and spinal cord ventral grey matter following nerve lesion will be used to identify novel candidate genes potentially involved in the critical CNTF receptor signaling. Relevance in lay language: We will determine how natural CNTF receptor signaling in mice protects and repairs the adult neuromuscular system. The resulting knowledge should facilitate the design of therapeutics which selectively enhance the appropriate CNTF receptor signaling and thereby effectively treat neuromuscular disease while avoiding side effects.
