The functional organization of axonal mRNA transport and translation.

Axons extend for tremendously long distances from the neuronal soma and make use of localized mRNA translation to rapidly respond to different extracellular stimuli and physiological states. The locally synthesized proteins support many different functions in both developing and mature axons, raising questions about the mechanisms by which local translation is organized to ensure the appropriate responses to specific stimuli. Publications over the past few years have uncovered new mechanisms for regulating the axonal transport and localized translation of mRNAs, with several of these pathways converging on the regulation of cohorts of functionally related mRNAs - known as RNA regulons - that drive axon growth, axon guidance, injury responses, axon survival and even axonal mitochondrial function. Recent advances point to these different regulatory pathways as organizing platforms that allow the axon's proteome to be modulated to meet its physiological needs.

Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits.

Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Axon Initial Segments Are Required for Efficient Motor Neuron Axon Regeneration and Functional Recovery of Synapses.

The axon initial segment (AIS) generates action potentials and maintains neuronal polarity by regulating the differential trafficking and distribution of proteins, transport vesicles, and organelles. Injury and disease can disrupt the AIS, and the subsequent loss of clustered ion channels and polarity mechanisms may alter neuronal excitability and function. However, the impact of AIS disruption on axon regeneration after injury is unknown. We generated male and female mice with AIS-deficient multipolar motor neurons by deleting AnkyrinG, the master scaffolding protein required for AIS assembly and maintenance. We found that after nerve crush, neuromuscular junction reinnervation was significantly delayed in AIS-deficient motor neurons compared with control mice. In contrast, loss of AnkyrinG from pseudo-unipolar sensory neurons did not impair axon regeneration into the intraepidermal nerve fiber layer. Even after AIS-deficient motor neurons reinnervated the neuromuscular junction, they failed to functionally recover because of reduced synaptic vesicle protein 2 at presynaptic terminals. In addition, mRNA trafficking was disrupted in AIS-deficient axons. Our results show that, after nerve injury, an intact AIS is essential for efficient regeneration and functional recovery of axons in multipolar motor neurons. Our results also suggest that loss of polarity in AIS-deficient motor neurons impairs the delivery of axonal proteins, mRNAs, and other cargoes necessary for regeneration. Thus, therapeutic strategies for axon regeneration must consider preservation or reassembly of the AIS.SIGNIFICANCE STATEMENT Disruption of the axon initial segment is a common event after nervous system injury. For multipolar motor neurons, we show that axon initial segments are essential for axon regeneration and functional recovery after injury. Our results may help explain injuries where axon regeneration fails, and suggest strategies to promote more efficient axon regeneration.

Analysis and interpretation of acylcarnitine profiles in dried blood spot and plasma of preterm and full-term newborns.

BACKGROUND: Acylcarnitines are biomarkers of fatty acid metabolism, and examining their patterns in preterm newborn may reveal metabolic changes associated with particular conditions related to prematurity. Isomeric acylcarnitines in dried blood spots (DBS) and plasma have never been assessed in preterm infants. METHODS: We studied 157 newborn divided into four groups by weeks of gestational age (GA), as follows: 22-27 wk in group 1; 28-31 wk in group 2; 32-36 wk in group 3; and 37-42 wk in group 4. Samples were collected on the third day of life. Acylcarnitines were separated and quantified using ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Acylcarnitine concentrations correlated significantly with GA and birth weight in both DBS and plasma samples. Concentrations were lower in preterm newborn, except for acylcarnitines derived from branched-chain amino acids, which were higher and correlated with enteral feeding. On day 3 of life, no correlations emerged with gender, respiratory distress syndrome, bronchopulmonary dysplasia, surfactant administration, or mechanical ventilation. CONCLUSION: We established GA-based reference ranges for isomeric acylcarnitine concentrations in preterm newborn, which could be used to assess nutritional status and the putative neuroprotective role of acylcarnitines.

Teneurin trans-axonal signaling prunes topographically missorted axons.

Building precise neural circuits necessitates the elimination of axonal projections that have inaccurately formed during development. Although axonal pruning is a selective process, how it is initiated and controlled in vivo remains unclear. Here, we show that trans-axonal signaling mediated by the cell surface molecules Glypican-3, Teneurin-3, and Latrophilin-3 prunes misrouted retinal axons in the visual system. Retinotopic neuron transplantations revealed that pioneer ventral axons that elongate first along the optic tract instruct the pruning of dorsal axons that missort in that region. Glypican-3 and Teneurin-3 are both selectively expressed by ventral retinal ganglion cells and cooperate for correcting missorted dorsal axons. The adhesion G-protein-coupled receptor Latrophilin-3 signals along dorsal axons to initiate the elimination of topographic sorting errors. Altogether, our findings show an essential function for Glypican-3, Teneurin-3, and Latrophilin-3 in topographic tract organization and demonstrate that axonal pruning can be initiated by signaling among axons themselves.

PTBP1 regulates injury responses and sensory pathways in adult peripheral neurons.

Polypyrimidine tract binding protein 1 (PTBP1) is thought to be expressed only at embryonic stages in central neurons. Its down-regulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently tested for generation of neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Unexpectedly, we found that both proteins are coexpressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 also shows axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and it associates in axons with mRNAs involved in injury responses and nerve regeneration, including importin ß1 (KPNB1) and RHOA. Perturbation of PTBP1 affects local translation in axons, nociceptor neuron regeneration and both thermal and mechanical sensation. Thus, PTBP1 has functional roles in adult axons. Hence, caution is required before considering targeting of PTBP1 for therapeutic purposes.

Emphysema lung lobe volume reduction: effects on the ipsilateral and contralateral lobes.

OBJECTIVES: To investigate volumetric and density changes in the ipsilateral and contralateral lobes following volume reduction of an emphysematous target lobe. METHODS: The study included 289 subjects with heterogeneous emphysema, who underwent bronchoscopic volume reduction of the most diseased lobe with endobronchial valves and 132 untreated controls. Lobar volume and low-attenuation relative area (RA) changes post-procedure were measured from computed tomography images. Regression analysis (Spearman's rho) was performed to test the association between change in the target lobe volume and changes in volume and density variables in the other lobes. RESULTS: The target lobe volume at full inspiration in the treatment group had a mean reduction of -0.45 L (SE = 0.034, P < 0.0001), and was associated with volume increases in the ipsilateral lobe (rho = -0.68, P < 0.0001) and contralateral lung (rho = -0.16, P = 0.006), and overall reductions in expiratory RA (rho = 0.31, P < 0.0001) and residual volume (RV)/total lung capacity (TLC) (rho = 0.13, P = 0.03). CONCLUSIONS: When the volume of an emphysematous target lobe is reduced, the volume is redistributed primarily to the ipsilateral lobe, with an overall reduction. Image-based changes in lobar volumes and densities indicate that target lobe volume reduction is associated with statistically significant overall reductions in air trapping, consistent with expansion of the healthier lung. KEY POINTS: Computed tomography allows assessment of the treatment of emphysema with endobronchial valves. • Endobronchial valves can reduce the volume of an emphysematous lung lobe. • Compensatory expansion is greater in ipsilateral lobes than in the contralateral lung. • Reduced air trapping is measurable by RV/TLC and smaller low attenuation area.

Neurobiology: Resetting the axon's batteries.

Neuronal injury can cause mitochondrial damage, leading to reduced energy production, decreased Ca2+ storage capacity, and increased reactive oxygen species. A new study reveals a mechanism to trigger the axonal transport of previously anchored mitochondria and promote neuroprotection and axon regeneration by replacing damaged with functional mitochondria.

Integration of Chest CT CAD into the Clinical Workflow and Impact on Radiologist Efficiency.

RATIONALE AND OBJECTIVES: The purpose of this paper is to describe the integration of a commercial chest CT computer-aided detection (CAD) system into the clinical radiology reporting workflow and perform an initial investigation of its impact on radiologist efficiency. It seeks to complement research into CAD sensitivity and specificity of stand-alone systems, by focusing on report generation time when the CAD is integrated into the clinical workflow. MATERIALS AND METHODS: A commercial chest CT CAD software that provides automated detection and measurement of lung nodules, ascending and descending aorta, and pleural effusion was integrated with a commercial radiology report dictation application. The CAD system automatically prepopulated a radiology report template, thus offering the potential for increased efficiency. The integrated system was evaluated using 40 scans from a publicly available lung nodule database. Each scan was read using two methods: (1) without CAD analytics, i.e., manually populated report with measurements using electronic calipers, and (2) with CAD analytics to prepopulate the report for reader review and editing. Three radiologists participated as readers in this study. RESULTS: CAD assistance reduced reading times by 7%-44%, relative to the conventional manual method, for the three radiologists from opening of the case to signing of the final report. CONCLUSION: This study provides an investigation of the impact of CAD and measurement on chest CTs within a clinical reporting workflow. Prepopulation of a report with automated nodule and aorta measurements yielded substantial time savings relative to manual measurement and entry.

Automatic segmentation of lung parenchyma in the presence of diseases based on curvature of ribs.

RATIONALE AND OBJECTIVES: Segmentation of lungs using high-resolution computer tomographic images in the setting of diffuse lung diseases is a major challenge in medical image analysis. Threshold-based techniques tend to leave out lung regions that have increased attenuation, such as in the presence of interstitial lung disease. In contrast, streak artifacts can cause the lung segmentation to "leak" into the chest wall. The purpose of this work was to perform segmentation of the lungs using a technique that selects an optimal threshold for a given patient by comparing the curvature of the lung boundary to that of the ribs. METHODS: Our automated technique goes beyond fixed threshold-based approaches to include lung boundary curvature features. One would expect the curvature of the ribs and the curvature of the lung boundary around the ribs to be very close. Initially, the ribs are segmented by applying a threshold algorithm followed by morphologic operations. The lung segmentation scheme uses a multithreshold iterative approach. The threshold value is verified until the curvature of the ribs and the curvature of the lung boundary are closely matched. The curve of the ribs is represented using polynomial interpolation, and the lung boundary is matched in such a way that there is minimal deviation from this representation. Performance of this technique was compared with conventional (fixed threshold) lung segmentation techniques on 25 subjects using a volumetric overlap fraction measure. RESULTS: The performance of the rib segmentation technique was significantly different from conventional techniques with an average higher mean volumetric overlap fraction of about 5%. CONCLUSIONS: The technique described here allows for accurate quantification of volumetric computed tomography and more advanced segmentation of abnormal areas.

CAD in clinical trials: current role and architectural requirements.

Computer-aided diagnosis (CAD) technology is becoming an important tool to assess treatment response in clinical trials. However, CAD software alone is not sufficient to conduct an imaging-based clinical trial. There are a number of architectural requirements such as image receive (from multiple field sites), a database for storing quantitative measures, and data mining and reporting capabilities. In this paper we describe the architectural requirements to incorporate CAD into clinical trials and illustrate their functionality in therapeutic trials for emphysema.

Reproducibility of lung and lobar volume measurements using computed tomography.

RATIONALE AND OBJECTIVES: Lung and lobar volume measurements from computed tomographic (CT) imaging are being used in clinical trials to assess new minimally invasive emphysema treatments aiming to reduce lung volumes. Establishing the reproducibility of lung volume measurements is important if they are to be accepted as treatment planning and outcome variables. The aims of this study were to (1) investigate the correlation between lung volumes assessed on CT imaging and on pulmonary function testing (PFT), (2) compare the two methods' reproducibility, and (3) assess the reproducibility of CT lobar volumes. MATERIALS AND METHODS: CT imaging and body plethysmography were performed at baseline and after a 9-month interval in multicenter emphysema treatment trials. Lung volumes were measured at total lung capacity (TLC) and at residual volume (RV). Lobar volumes were measured on CT imaging using a semiautomated technique. The correlations between CT and PFT volumes were computed for 486 subjects at baseline. Reproducibility was assessed in terms of the intraclass correlation coefficient (ICC) for 126 subjects from the control group at TLC and 120 subjects at RV. RESULTS: Correlations between CT and PFT lung volumes were 0.86 at TLC and 0.67 at RV. At TLC, the ICCs were 0.943 for CT imaging and 0.814 for PFT. At RV, the ICCs were 0.886 for CT imaging and 0.683 for PFT. CT lobar volumes showed good reproducibility (all P values < .05). CONCLUSION: CT lung and lobar volume measurements could be captured in a multicenter trial setting with high reproducibility and were highly correlated with those obtained on PFT. CT imaging showed significantly better reproducibility than PFT between interval lung volume measurements, offering the potential for designing emphysema treatment trials involving fewer subjects.