Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Undirected singing rate as a non-invasive tool for welfare monitoring in isolated male zebra finches.

Research on the songbird zebra finch (Taeniopygia guttata) has advanced our behavioral, hormonal, neuronal, and genetic understanding of vocal learning. However, little is known about the impact of typical experimental manipulations on the welfare of these birds. Here we explore whether the undirected singing rate can be used as an indicator of welfare. We tested this idea by performing a post hoc analysis of singing behavior in isolated male zebra finches subjected to interactive white noise, to surgery, or to tethering. We find that the latter two experimental manipulations transiently but reliably decreased singing rates. By contraposition, we infer that a high-sustained singing rate is suggestive of successful coping or improved welfare in these experiments. Our analysis across more than 300 days of song data suggests that a singing rate above a threshold of several hundred song motifs per day implies an absence of an acute stressor or a successful coping with stress. Because singing rate can be measured in a completely automatic fashion, its observation can help to reduce experimenter bias in welfare monitoring. Because singing rate measurements are non-invasive, we expect this study to contribute to the refinement of the current welfare monitoring tools in zebra finches.

Hippocampal Remapping after Partial Inactivation of the Medial Entorhinal Cortex.

Hippocampal place cells undergo remapping when the environment is changed. The mechanism of hippocampal remapping remains elusive but spatially modulated cells in the medial entorhinal cortex (MEC) have been identified as a possible contributor. Using pharmacogenetic and optogenetic approaches, we tested the role of MEC cells by examining in mice whether partial inactivation in MEC shifts hippocampal activity to a different subset of place cells with different receptive fields. The pharmacologically selective designer Gi-protein-coupled muscarinic receptor hM4D or the light-responsive microbial proton pump archaerhodopsin (ArchT) was expressed in MEC, and place cells were recorded after application of the inert ligand clozapine-N-oxide (CNO) or light at appropriate wavelengths. CNO or light caused partial inactivation of the MEC. The inactivation was followed by substantial remapping in the hippocampus, without disruption of the spatial firing properties of individual neurons. The results point to MEC input as an element of the mechanism for remapping in place cells.

Large-scale axonal reorganization of inhibitory neurons following retinal lesions.

The functional properties of adult cortical neurons are subject to alterations in sensory experience. Retinal lesions lead to remapping of cortical topography in the region of primary visual cortex representing the lesioned part of the retina, the lesion projection zone (LPZ), with receptive fields shifting to the intact parts of the retina. Neurons within the LPZ receive strengthened input from the surrounding region by growth of the plexus of excitatory long-range horizontal connections. Here, by combining cell type-specific labeling with a genetically engineered recombinant adeno-associated virus and in vivo two-photon microscopy in adult macaques, we showed that the remapping was also associated with alterations in the axonal arbors of inhibitory neurons, which underwent a parallel process of pruning and growth. The axons of inhibitory neurons located within the LPZ extended across the LPZ border, suggesting a mechanism by which new excitatory input arising from the peri-LPZ is balanced by reciprocal inhibition arising from the LPZ.

Map fragmentation in two- and three-dimensional environments.

The suggestion that three-dimensional space is represented by a mosaic of neural map fragments, each covering a small area of space in the plane of locomotion, receives support from studies in complex two-dimensional environments. How map fragments are linked, which brain circuits are involved, and whether metric is preserved across fragments are questions that remain to be determined.

Axonal dynamics of excitatory and inhibitory neurons in somatosensory cortex.

Cortical topography can be remapped as a consequence of sensory deprivation, suggesting that cortical circuits are continually modified by experience. To see the effect of altered sensory experience on specific components of cortical circuits, we imaged neurons, labeled with a genetically modified adeno-associated virus, in the intact mouse somatosensory cortex before and after whisker plucking. Following whisker plucking we observed massive and rapid reorganization of the axons of both excitatory and inhibitory neurons, accompanied by a transient increase in bouton density. For horizontally projecting axons of excitatory neurons there was a net increase in axonal projections from the non-deprived whisker barrel columns into the deprived barrel columns. The axon collaterals of inhibitory neurons located in the deprived whisker barrel columns retracted in the vicinity of their somata and sprouted long-range projections beyond their normal reach towards the non-deprived whisker barrel columns. These results suggest that alterations in the balance of excitation and inhibition in deprived and non-deprived barrel columns underlie the topographic remapping associated with sensory deprivation.

Experience-dependent gene expression in adult visual cortex.

Experience-dependent plasticity of the adult visual cortex underlies perceptual learning and recovery of function following central nervous system lesions. To reveal the signal transduction cascades involved in adult cortical plasticity, we utilized a model of remapping of cortical topography following binocular retinal lesions. In this model, the lesion projection zone (LPZ) of primary visual cortex (V1) recovers visually driven activity by the sprouting of horizontal axonal connections originating from the cells in the surrounding region. To explore the molecular mechanism underlying this process, we used gene microarrays from an expression library prepared from Macaque V1. By microarray analysis of gene expression levels in the LPZ and the surrounding region, and subsequent confirmation with Quantitative Real-Time polymerase chain reaction and in situ hybridization, the participation of a number of genes was observed, including the Rho GTPase family. Its role in regulation of cytoskeleton assembly provides a possible link between the alteration of neural activity and cortical functional reorganization.

Rapid axonal sprouting and pruning accompany functional reorganization in primary visual cortex.

The functional architecture of adult cerebral cortex retains a capacity for experience-dependent change. This is seen following focal binocular lesions, which induce rapid changes in receptive field size and position. To follow the dynamics of the circuitry underlying these changes, we imaged the intrinsic long-range horizontal connections within the lesion projection zone (LPZ) in adult macaque primary visual cortex. To image the same axons over time, we combined viral vector-mediated EGFP transfer and two-photon microscopy. The lesion triggered, within the first week, an approximately 2-fold outgrowth of axons toward the center of the LPZ. Over the subsequent month, axonal density declined due to a parallel process of pruning and sprouting but maintained a net increase relative to prelesion levels. The rate of turnover of axonal boutons also increased. The axonal restructuring recapitulates the pattern of exuberance and pruning seen in early development and correlates well with the functional changes following retinal lesions.

Axons and synaptic boutons are highly dynamic in adult visual cortex.

While recent studies of synaptic stability in adult cerebral cortex have focused on dendrites, how much axons change is unknown. We have used advances in axon labeling by viruses and in vivo two-photon microscopy to investigate axon branching and bouton dynamics in primary visual cortex (V1) of adult Macaque monkeys. A nonreplicative adeno-associated virus bearing the gene for enhanced green fluorescent protein (AAV.EGFP) provided persistent labeling of axons, and a custom-designed two-photon microscope enabled repeated imaging of the intact brain over several weeks. We found that large-scale branching patterns were stable but that a subset of small branches associated with terminaux boutons, as well as a subset of en passant boutons, appeared and disappeared every week. Bouton losses and gains were both approximately 7% of the total population per week, with no net change in the overall density. These results suggest ongoing processes of synaptogenesis and elimination in adult V1.