Abstracts

Rationale: RNA-binding proteins (RBPs) are emerging as causal agents of complex neurological diseases, including epilepsy. Mice deficient for the neuronal RBP CELF4 have a complex seizure disorder, are hyperactive, and have late-onset obesity. Recently, human CELF4 was associated with clinical features similar to that of mutant mice, indicating that CELF4 is important for mammalian neurological function. We previously showed that CELF4 is expressed mainly in excitatory neurons of the cerebral cortex and hippocampus and that it regulates excitatory, but not inhibitory, neurotransmission. Methods: Here we further examine the mechanism underlying neuronal excitability and seizures of Celf4 mutants by identifying CELF4 target mRNAs using iCLIP, assessing their fate in Celf4 mutants using transcriptome analysis and validation by qPCR and immunostaining, and evaluating neuronal function in Celf4 mutant mice with electrophysiology. Results: We find that CELF4 selectively binds 15-20% of the transcriptome, with striking enrichment for the 3' UTR. Many CELF4-bound mRNAs encode proteins involved in neuron development and function. Global changes in steady-state levels of CELF4 targets are generally modest, however transcriptome analysis of polysome fractions as well as cell body-neuropil dissections suggests CELF4 is critical for maintaining mRNA stability and availability for translation. Patch-clamp recordings of wildtype and Celf4 mutant cortical neurons revealed that Celf4 heterozygotes and homozygotes are more likely to generate action potentials (p<0.01) and have higher persistent sodium current (I_NaP, p<0.01), for which SCN8A (Na_v1.6) is a major determinant. Indeed, Scn8a mRNA is a direct CELF4 target, and immunostaining showed a dramatic increase in SCN8A level at the axon initial segment of Celf4 mutants (p<0.0001). This increase appears to be critical for seizure generation in Celf4 mutant mice, as lowering the gene dosage of normal Scn8a leads to a remarkable elevation of seizure threshold in Celf4 mutants (p<0.001). Last, Celf4 mutant neurons display diminished homeostatic plasticity following bicuculline treatment, suggesting that the overarching role of CELF4 may be as an important regulator of homeostasis in excitatory neurons. Conclusions: Overall, these results strongly suggest that dysregulation of CELF4 target mRNAs increases neuronal excitability - at least in part by upregulating SCN8A at the AIS - and impairs homeostatic response, together leading to a complex seizure disorder. Significantly, these studies support and expand the vital roles RBPs play in regulating and fine-tuning activity of neural circuits and highlight the importance of RBP function in neurological disease.