Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

Effects of Transcriptional Pausing on Gene Expression Dynamics

Stochasticity in gene expression affects many cellular processes and is a source of phenotypic diversity between genetically identical individuals. Events in elongation, particularly RNA polymerase pausing, are a source of this noise. Since the rate and duration of pausing are sequence-dependent, this regulatory mechanism of transcriptional dynamics is evolvable. The dependency of pause propensity on regulatory molecules makes pausing a response mechanism to external stress. Using a delayed stochastic model of bacterial transcription at the single nucleotide level that includes the promoter open complex formation, pausing, arrest, misincorporation and editing, pyrophosphorolysis, and premature termination, we investigate how RNA polymerase pausing affects a gene's transcriptional dynamics and gene networks. We show that pauses' duration and rate of occurrence affect the bursting in RNA production, transcriptional and translational noise, and the transient to reach mean RNA and protein levels. In a genetic repressilator, increasing the pausing rate and the duration of pausing events increases the period length but does not affect the robustness of the periodicity. We conclude that RNA polymerase pausing might be an important evolvable feature of genetic networks

Spatial Stereoresolution for Depth Corrugations May Be Set in Primary Visual Cortex

Stereo “3D” depth perception requires the visual system to extract binocular disparities between the two eyes' images. Several current models of this process, based on the known physiology of primary visual cortex (V1), do this by computing a piecewise-frontoparallel local cross-correlation between the left and right eye's images. The size of the “window” within which detectors examine the local cross-correlation corresponds to the receptive field size of V1 neurons. This basic model has successfully captured many aspects of human depth perception. In particular, it accounts for the low human stereoresolution for sinusoidal depth corrugations, suggesting that the limit on stereoresolution may be set in primary visual cortex. An important feature of the model, reflecting a key property of V1 neurons, is that the initial disparity encoding is performed by detectors tuned to locally uniform patches of disparity. Such detectors respond better to square-wave depth corrugations, since these are locally flat, than to sinusoidal corrugations which are slanted almost everywhere. Consequently, for any given window size, current models predict better performance for square-wave disparity corrugations than for sine-wave corrugations at high amplitudes. We have recently shown that this prediction is not borne out: humans perform no better with square-wave than with sine-wave corrugations, even at high amplitudes. The failure of this prediction raised the question of whether stereoresolution may actually be set at later stages of cortical processing, perhaps involving neurons tuned to disparity slant or curvature. Here we extend the local cross-correlation model to include existing physiological and psychophysical evidence indicating that larger disparities are detected by neurons with larger receptive fields (a size/disparity correlation). We show that this simple modification succeeds in reconciling the model with human results, confirming that stereoresolution for disparity gratings may indeed be limited by the size of receptive fields in primary visual cortex

A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria

Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods

Sortilin Participates in Light-dependent Photoreceptor Degeneration in Vivo

Both proNGF and the neurotrophin receptor p75 (p75NTR) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75NTR co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75NTR, and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75NTR and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the “pro” domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75NTR/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies

NGF and proNGF Regulate Functionally Distinct mRNAs in PC12 Cells: An Early Gene Expression Profiling

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a “less potent NGF” is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a “pure proNGF” signalling, distinct from the “pure NGF” one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs

Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1

Small molecule activators of the Trk receptors for neuroprotection

The neurotophin signaling network is critical to the development and survival of many neuronal populations. Especially sensitive to imbalances in the neurotrophin system, cholinergic neurons in the basal forebrain are progressively lost in Alzheimer's disease. Therapeutic use of neurotrophins to prevent this loss is hampered, however, by a number of pharmacological challenges. These include a lack of transport across the blood-brain barrier, rapid degradation in the circulation, and difficulty in production. In this review we discuss the evidence supporting the neurotrophin system's role in preventing neurodegeneration and survey some of the pharmacological strategies being pursued to develop effective therapeutics targeting neurotrophin function

A putative functional role for oligodendrocytes in mood regulation

Altered glial structure and function is implicated in several major mental illnesses and increasing evidence specifically links changes in oligodendrocytes with disrupted mood regulation. Low density and reduced expression of oligodendrocyte-specific gene transcripts in postmortem human subjects points toward decreased oligodendrocyte function in most of the major mental illnesses. Similar features are observed in rodent models of stress-induced depressive-like phenotypes, such as the unpredictable chronic mild stress and chronic corticosterone exposure, suggesting an effect downstream from stress. However, whether oligodendrocyte changes are a causal component of psychiatric phenotypes is not known. Traditional views that identify oligodendrocytes solely as nonfunctional support cells are being challenged, and recent studies suggest a more dynamic role for oligodendrocytes in neuronal functioning than previously considered, with the region adjacent to the node of Ranvier (i.e., paranode) considered a critical region of glial–neuronal interaction. Here, we briefly review the current knowledge regarding oligodendrocyte disruptions in psychiatric disorders and related animal models, with a focus on major depression. We then highlight several rodent studies, which suggest that alterations in oligodendrocyte structure and function can produce behavioral changes that are informative of mood regulatory mechanisms. Together, these studies suggest a model, whereby impaired oligodendrocyte and possibly paranode structure and function can impact neural circuitry, leading to downstream effects related to emotionality in rodents, and potentially to mood regulation in human psychiatric disorders