Mechanisms underlying synapse development are fundamental in shaping functional neural circuitry, while impairments in these mechanisms contribute to many nervous system diseases. The neuromuscular junction (NMJ) has been extensively studied as a model synapse, as it is large and readily accessible. Synaptic enrichment of mRNAs encoding NMJ components is achieved through transcriptional upregulation in subsynaptic nuclei. Synaptically enriched mRNAs are likely to be involved in synapse formation or function;one such mRNA, 7H4, is a noncoding RNA whose synapse-associated and neurally controlled expression is consistent with a role in postnatal NMJ development. Interestingly, embedded in 7H4 is the sequence of a microRNA (miRNA), miR-133b. MiRNAs provide widespread regulation of gene expression through silencing of target mRNAs, however, their role in synaptic development is unclear. Postnatal, synaptic enrichment of miR-133b may cause silencing of key target mRNAs at the NMJ, which could be required for appropriate development and maturation of the NMJ. This proposal seeks to uncover a role for miRNAs in synapse development, using the NMJ as a model synapse.
The first aim will establish whether the unique expression pattern of 7H4 is reflected at the level of miR-133b. MiRNA northern blots and in situ hybridization have established synaptic and postnatal enrichment of miR-133b, while neural regulation will be explored by miRNA northern blot following denervation of hindlimb muscle.
The second aim will determine whether miR-133b is critical for NMJ formation. miR-133b will be overexpressed or knocked down in cultured myotubes and muscle, with subsequent analysis of synapse development. Synaptic phenotypes will also be assessed in mice with targeted deletion of miR-133b.
The third aim seeks to elucidate the mechanism of action of miR-133b by determining its mRNA targets. Using computational target prediction and luciferase reporter assays, glial cell line derived neurotrophic factor (GDNF) has been validated as an in vitro target for miR-133b. Silencing of endogenous GDNF will be examined in cultured glial cells, and GDNF will be validated as a target in vivo if miR-133b knockout leads to its upregulation. Together, these strategies will aid the identification of novel miRNA-mediated mechanisms in NMJ development, and may provide insights into devastating NMJ disorders such as congenital myasthenic syndromes. This work will deepen our understanding of the complexities of synapse development, with future relevance for central synaptic disorders, which are of significant concern for public health.
|Heyer, Mary P; Pani, Amar K; Smeyne, Richard J et al. (2012) Normal midbrain dopaminergic neuron development and function in miR-133b mutant mice. J Neurosci 32:10887-94|