Nicholas J. Hornstein

In recent years, a number of tools have become available for remotely activating neural circuits in Drosophila. Despite widespread and growing use, very little work has been done to characterize exactly how these tools affect activity in identified fly neurons. Using the GAL4-UAS system, we expressed blue light-gated Channelrhodopsin-2 (ChR2) and a mutated form of ChR2 (H134R-ChR2) in motor and sensory neurons of the Drosophila third-instar locomotor circuit. Neurons expressing H134R-ChR2 show enhanced responses to blue light pulses and less spike frequency adaptation than neurons expressing ChR2. Although H134R-ChR2 was more effective at manipulating behavior than ChR2, the behavioral consequences of firing rate adaptation were different in sensory and motor neurons. For comparison, we examined the effects of ectopic expression of the warmth-activated cation channel Drosophila TRPA1 (dTRPA1). When dTRPA1 was expressed in larval motor neurons, heat ramps from 21 to 27 degrees C evoked tonic spiking at approximately 25 degrees C that showed little adaptation over many minutes. dTRPA1 activation had stronger and longer-lasting effects on behavior than ChR2 variants. These results suggest that dTRPA1 may be particularly useful for researchers interested in activating fly neural circuits over long time scales. Overall, this work suggests that understanding the cellular effects of these genetic tools and their temporal dynamics is important for the design and interpretation of behavioral experiments.

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5'-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation.

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome.

Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.