BAC-trap studies of Purkinje cells in normal and FMR1 mutant mice

The molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) are limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to riboCGG repeat-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA binding proteins involved in translation that aberrantly interact with the toxic RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of riboCGG-induced phenotypes. This approach identified nearly 500 transcripts that are differentially associated with ribosomes in r(CGG)90-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)90 list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)90 repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)90expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest translational dysregulation may be an underlying mechanism of riboCGG-induced neurotoxicity and provide insight into the pathogenicity of FXTASBAC-trap studies of Purkinje cels in normal and mutant mice

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Galloway JN, Shaw C, Yu P, Parghi D, Poidevin M, Jin P, Nelson DL