Completion of neuronal remodeling prompts myelination along developing motor axon branches.

Neuronal remodeling and myelination are two fundamental processes during neurodevelopment. How they influence each other remains largely unknown, even though their coordinated execution is critical for circuit function and often disrupted in neuropsychiatric disorders. It is unclear whether myelination stabilizes axon branches during remodeling or whether ongoing remodeling delays myelination. By modulating synaptic transmission, cytoskeletal dynamics, and axonal transport in mouse motor axons, we show that local axon remodeling delays myelination onset and node formation. Conversely, glial differentiation does not determine the outcome of axon remodeling. Delayed myelination is not due to a limited supply of structural components of the axon-glial unit but rather is triggered by increased transport of signaling factors that initiate myelination, such as neuregulin. Further, transport of promyelinating signals is regulated via local cytoskeletal maturation related to activity-dependent competition. Our study reveals an axon branch-specific fine-tuning mechanism that locally coordinates axon remodeling and myelination.

Trajectory data of antero- and retrograde movement of mitochondria in living zebrafish larvae.

Recently, a large number of single particle tracking (SPT) approaches have been developed. Generally, SPT techniques can be split into two groups: ex post facto approaches where trajectory extraction is carried out after data acquisition and feedback based approaches that perform particle tracking in real time [1]. One feedback approach is 3D Orbital Tracking, where the laser excitation beam is rotated in a circle about the object, generating a so called orbit [2,3]. By calculating the particle position from the detected intensity after every orbit in relation to its center, this method allows the microscope to follow a single object in real time. The high spatiotemporal resolution of this method and the potential to optically manipulate the followed object during the measurement promises to yield new deep insights into biological systems [4-7]. By upgrading this approach in a way that the specimen is recentered by a xy-stage on the center of the microscope, particle tracking with this long-range tracking feature is no longer limited to the covered field-of-view. This allows for the observation of mitochondrial trafficking in living zebrafish embryos over long distances. Here, we provide the raw data for antero- and retrograde movement of mitochondria labelled with photo-activatable green fluorescent protein (mitoPAGFP). It relates to the scientific article "Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo" [8]. By applying a correlation analysis on the trajectories, it is possible to distinguish between active transport and pausing events with less biasing compared to the mean squared displacement approach.

Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo.

We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resolution. By combining various technical improvements, we tracked individual mitochondria with unprecedented spatiotemporal resolution over distances of >100 µm. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resolution in a living organism offers a new approach to elucidating their function in its complete physiological context.

Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca2+/CaM Controls DCV Binding and Liprin-α/TANC2 Recruits DCVs to Postsynaptic Sites.

Tight regulation of neuronal transport allows for cargo binding and release at specific cellular locations. The mechanisms by which motor proteins are loaded on vesicles and how cargoes are captured at appropriate sites remain unclear. To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2. We showed that calcium, acting via CaM, enhances KIF1A binding to DCVs and increases vesicle motility. In contrast, liprin-α and TANC2 are not part of the KIF1A-cargo complex but capture DCVs at dendritic spines. Furthermore, we found that specific TANC2 mutations-reported in patients with different neuropsychiatric disorders-abolish the interaction with KIF1A. We propose a model in which Ca2+/CaM regulates cargo binding and liprin-α and TANC2 recruit KIF1A-transported vesicles.

Field proteomics of Populus alba grown in a heavily modified environment - An example of a tannery waste landfill.

Tannery waste is highly toxic and dangerous to living organisms because of the high heavy metal content, especially chromium [Cr(III)]. This study analysed the proteomic response of the Populus alba L. clone 'Villafranca' grown for 4years on a tannery waste landfill. In this extremely hostile environment, the plants struggled with continuous stress, which inhibited growth by 54%, with a 67% decrease in tree height and diameter at breast height compared to those of the forest reference plot, respectively. The leaves and roots of the tannery landfill-grown plants produced strong proteomic stress signals for protection against reactive oxygen species (ROS) and repair to ROS-damaged proteins and DNA as well as signals for protection of the photosynthetic apparatus. The content of HSP80 was also high. However, primary metabolic pathways were generally unaffected, and signals of increased protein protection, but not turnover, were found, indicating mechanisms of adaptation to long-term stress conditions present at the landfill. A proteomic tool, two-dimensional electrophoresis coupled with tandem mass spectrometry, was successfully applied in this environmental in situ study of distant plots (280km apart).

Imaging of neuronal mitochondria in situ.

Neuronal mitochondria are receiving a rapidly increasing level of attention. This is to a significant part due to the ability to visualize neuronal mitochondria in novel ways, especially in vivo. Such an approach allows studying neuronal mitochondria in an intact tissue context, during different developmental states and in various genetic backgrounds and disease conditions. Hence, in vivo imaging of mitochondria in the nervous system can reveal aspects of the 'mitochondrial life cycle' in neurons that hitherto have remained obscure or could only be inferred indirectly. In this survey of the current literature, we review the new insights that have emerged from studies using mitochondrial imaging in intact neural preparations ranging from worms to mice.

Response of leaf and fine roots proteomes of Salix viminalis L. to growth on Cr-rich tannery waste.

The tannery industry is a major source of anthropogenic chromium (Cr) contamination due to the large amounts of solid waste produced and its problematic management. The unique composition of tannery waste, usually high concentrations of Cr and other metals as well as organic matter and nutrients, makes it a great risk for soil and water environment but also a possible effective fertilizer for non-food plants that can tolerate metals. The goal of this study was to understand the adaptation mechanism of Salix viminalis to growth on Cr-rich tannery waste from an active landfill. We used a proteomic approach to identify leaf and fine roots proteins altered by tannery waste as compared to control soil conditions. We found no obvious symptoms of oxidative stress in leaves or fine roots. Proteomic results indicated some changes in metabolism, with increases in energy production processes and their greater efficiency for leaves rather than root development. Comparison between S. viminalis and P. × canescens response to tannery waste suggested that S. viminalis is not suitable for remediation of Cr-contaminated areas of a tannery waste landfill site.

Imaging Subcellular Structures in the Living Zebrafish Embryo.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Utilization of proteomics in experimental field conditions--A case study of poplars growing on grassland affected by long-term starch wastewater irrigation.

The presented study verified the possibility of using proteomics as a tool for investigating poplars growing on obviously separate plots. The examination covered poplars planted on grassland irrigated for 40 years with potato industry wastewater and in a plot appropriate for poplar planting, spaced at a distance of 67 km from each other (hereinafter referred to as forest). The work aimed to compare the obtained proteomic results with data on biometric and biochemical parameters and mineral composition as well as to assess, at a molecular level, the usefulness of grasslands for planting. Proteome analysis showed that most of the stress-related proteins detected were less abundant on the irrigated grassland, confirming the viability of its revegetation with poplars. Proteomic data corresponded well with the other results, highlighting the probable reason for the proteome changes; i.e. deficiency of phosphate ions detected in the forest area. Moreover, proteome analysis revealed biotic stress symptoms in plants growing on the grassland, which were also well explained by other data but would not have been detected without performing the proteomic analysis. Therefore, environmental plant proteomics is a useful and valuable tool during field studies, even when samples are taken from plots some distance apart.

Epileptogenesis following Kainic Acid-Induced Status Epilepticus in Cyclin D2 Knock-Out Mice with Diminished Adult Neurogenesis.

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2-10 days) in wt mice and 8 days (range 2-16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1-3.4; cD2 KO: 0.57, range 0.1-2.0 seizures/day) or median seizure duration (wt: 51 s, range 23-103; cD2 KO: 51 s, range 23-103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state.

Populus × canescens grown on Cr-rich tannery waste: Comparison of leaf and root biochemical and proteomic responses.

Treatment of tannery effluents generates large amounts of sediments containing concentrated doses of metals (mainly chromium). Such waste is most commonly disposed of by landfilling, which is hazardous to the ecosystem due to Cr leaching. Afforestation of disposal sites with fast growing trees could stabilize contaminants in the soil and prevent them from spreading. The aim of this study was to examine the adaptation of Populus × canescens Sm. to tannery waste using biochemical and proteomic methods. We analyzed changes in the leaves and fine roots of poplar planted in soil or tannery waste. We found no obvious symptoms of metal stress, such as: elevated hydrogen peroxide levels or lipid peroxidation, but we observed activation of many elements of antioxidative system. Comparison of 2-DE protein profiles of leaves and fine roots from poplar grown on soil or tannery waste revealed increased expression of glycolytic enzymes and proteins involved in the synthesis of cell wall components, changes in the levels of proteins associated with photosynthesis, stress-related proteins, proteasome subunits and methionine biosynthesis enzymes. This experiment demonstrated that proteomic analysis has the potential to link the effects of Cr-rich tannery waste with biological consequences.

In vivo imaging of mitochondria in intact zebrafish larvae.

Visualizing neuronal mitochondria in a living, intact mammalian organism is a challenge that can be overcome in zebrafish larvae, which are highly accessible for optical imaging and genetic manipulation. Here, we detail an approach to visualize neuronal mitochondria in sensory Rohon-Beard axons, which allows quantitatively measuring mitochondrial shape, dynamics, and transport in vivo. This provides a useful assay for basic studies exploring the behavior of neuronal mitochondria in their natural habitat, for revealing the influence that disease-related alterations have on this behavior and for testing pharmacological compounds and genetic manipulations that might ameliorate disease-related mitochondrial phenotypes in neurons.

An optimized method to extract poplar leaf proteins for two-dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds.

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High-quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, "Eridano," "Villafranca," "NE-42," and "Luisa Avanzo," which have not yet been used for the proteomic studies.

An optimized method to extract poplar leaf proteins for two-dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds.

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of TCA and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High-quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method (OPTI) for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars i.e. 'Eridano', 'Villafranca', 'NE-42' and 'Luisa Avanzo', which have not yet been used for the proteomic studies.

In vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system.

Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.

Post-treatment with rapamycin does not prevent epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy.

Approximately 30% of all epilepsy cases are acquired. At present there is no effective strategy to stop epilepsy development after the precipitating insult. Recent data from experimental models pointed to the mTOR pathway, which can be potently inhibited by rapamycin. However, data on the antiepileptic and antiepileptogenic properties of rapamycin are conflicting. Therefore, we tested whether rapamycin post-treatment influences epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy in rats. Animals were treated with rapamycin (6mg/kg) or vehicle daily for 2 wks, beginning 24h after stimulation. Sham-operated animals were treated with rapamycin or vehicle but were not stimulated. Animals were video-EEG monitored to detect spontaneous seizures. Animals were sacrificed 4 wks later and brains were collected for Timm staining. There were no significant differences in the number of stimulated rats developing epilepsy; latency to first spontaneous seizure; number of seizures, or seizure frequency in epileptic animals. The area occupied by mossy fibers was significantly increased in stimulated vs. sham-operated animals but was not different in animals treated with rapamycin vs. vehicle. Collectively, our data suggest that the antiepileptic or antiepileptogenic action of rapamycin is not a universal phenomenon and might be limited to certain experimental models or experimental conditions.

Status epilepticus evokes prolonged increase in the expression of CCL3 and CCL4 mRNA and protein in the rat brain.

CCL3 and CCL4 are proinflammatory chemokines belonging to the CC family. Increase in expression of mRNA coding for various chemokines including CCL3 and CCL4 has been often detected with global transcriptome profiling of brain tissue following epileptogenic stimuli as well as in epilepsy in experimental models and in human patients. Despite this, little is known about the expression of these proteins in epileptogenesis or epilepsy. In the present work CCL3 and CCL4 mRNA and protein expression were studied in the amygdala stimulation model of temporal lobe epilepsy using quantitiative PCR and immunohistochemistry. Expression of CCL3 and CCL4 mRNA in the block of tissue containing enthorinal and piriform cortices, amygdala and piriform nucleus was markedly up-regulated at 1, 4, 14 and 30 days following stimulation and in hippocampal CA1 was significantly increased at 1 and 4 days following stimulation. Expression of CCL3 and CCL4 proteins was elevated in astrocytes in the enthorinal and piriform cortices, amygdala, and hippocampus showing the largest increase at 4D after status epilepticus. Increase in mRNA and protein levels of CCL3 and CCL4 in the animal model of temporal lobe epilepsy suggests their role in disease development or recovery form epileptogenic insult. Existence of multiple targets for these chemokines in the damaged brain allows several possibilities of influencing neuronal and glial functions.

Thresholds of heavy-metal toxicity in cuttings of European black poplar (Populus nigra L.) determined according to antioxidant status of fine roots and morphometrical disorders.

The effects of increasing concentrations of polluted soils collected from two different stands in the direct neighbourhood of a copper smelter were studied by analyzing the activity of lipid peroxidation and antioxidant enzymes in the fine roots of cuttings of black poplar (Populus nigra L.). Morphological parameters of affected and unaffected plants were compared and concentrations of heavy metals in their fine roots were estimated. Copper and lead were the major pollutants, and their threshold concentrations were determined on the basis of positive or negative impact on shoots and root growth, morphological disorders of the roots (thickening, decreasing of fine roots volume), stimulation or inhibition of the most important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), guaiacol and ascorbate peroxidases (GPOX, APOX) and glutathione reductase (GR) as well as increasing MDA concentration. Finally, three thresholds of Cu and Pb concentrations resulting in increasing toxicity against plants were proposed, splitting the ranges of the non toxic presence of these metals into soil (Cu<100 ppm, Pb<30 ppm), full tolerance (Cu 100-220 ppm, Pb 30-70 ppm), limited tolerance (Cu 220-650 ppm, Pb 70-200 ppm) and breakdown of tolerance (Cu>650 ppm, Pb>200 ppm).

Changes in carbohydrate metabolism in fine roots of the native European black poplar (Populus nigra L.) in a heavy-metal-polluted environment.

Effects of copper-smelter-related deposition of heavy metals in the soil on carbohydrate metabolism of fine roots of the native European black poplar were investigated in spring and autumn. Total soluble non-structural carbohydrates in fine roots from trees growing in the polluted habitat were lower than in a control site, but this was directly associated only with a lower raffinose concentration. Neither glucose nor fructose concentrations differed significantly between polluted and unpolluted sites. In contrast, the galactose concentration was higher in the presence of heavy metals, especially in autumn. Also the stachyose concentration was higher in the polluted site, but only in autumn, suggesting it could be an alternative way of detoxification of galactose. No difference between control and polluted stands was observed in sucrose concentration. However, estimates of sucrolytic activity revealed markedly higher activities of sucrose synthase (SuSy), soluble acid (AI) and neutral (NI) invertases in the polluted stand than in the control. In contrast, the estimated glycolytic enzyme activities were not affected by the presence of heavy metals in soil.