Neuronal Redevelopment and the Regeneration of Neuromodulatory Axons in the Adult Mammalian Central Nervous System.

One reason that many central nervous system injuries, including those arising from traumatic brain injury, spinal cord injury, and stroke, have limited recovery of function is that neurons within the adult mammalian CNS lack the ability to regenerate their axons following trauma. This stands in contrast to neurons of the adult mammalian peripheral nervous system (PNS). New evidence, provided by single-cell expression profiling, suggests that, following injury, both mammalian central and peripheral neurons can revert to an embryonic-like growth state which is permissive for axon regeneration. This "redevelopment" strategy could both facilitate a damage response necessary to isolate and repair the acute damage from injury and provide the intracellular machinery necessary for axon regrowth. Interestingly, serotonin neurons of the rostral group of raphe nuclei, which project their axons into the forebrain, display a robust ability to regenerate their axons unaided, counter to the widely held view that CNS axons cannot regenerate without experimental intervention after injury. Furthermore, initial evidence suggests that norepinephrine neurons within the locus coeruleus possess similar regenerative abilities. Several morphological characteristics of serotonin axon regeneration in adult mammals, observable using longitudinal in vivo imaging, are distinct from the known characteristics of unaided peripheral nerve regeneration, or of the regeneration seen in the spinal cord and optic nerve that occurs with experimental intervention. These results suggest that there is an alternative CNS program for axon regeneration that likely differs from that displayed by the PNS.

Tanycyte ablation in the arcuate nucleus and median eminence increases obesity susceptibility by increasing body fat content in male mice.

Tanycytes are radial glial cells located in the mediobasal hypothalamus. Recent studies have proposed that tanycytes play an important role in hypothalamic control of energy homeostasis, although this has not been directly tested. Here, we report the phenotype of mice in which tanycytes of the arcuate nucleus and median eminence were conditionally ablated in adult mice. Although the cerebrospinal fluid-hypothalamic barrier was rendered more permeable following tanycyte ablation, neither the blood-hypothalamic barrier nor leptin-induced pSTAT3 activation in hypothalamic parenchyma were affected. We observed a significant increase in visceral fat distribution accompanying insulin insensitivity in male mice, without significant effect on either body weight or food intake. A high-fat diet tended to accelerate overall body weight gain in tanycyte-ablated mice, but the development of visceral adiposity and insulin insensitivity was comparable to wildtype. Thermoneutral housing exacerbated fat accumulation and produced a shift away from fat oxidation in tanycyte-ablated mice. These results clarify the extent to which tanycytes regulate energy balance, and demonstrate a role for tanycytes in regulating fat metabolism.

Qdot nanobarcodes for multiplexed gene expression analysis.

We report a quantum dot (Qdot) nanobarcode-based microbead random array platform for accurate and reproducible gene expression profiling in a high-throughput and multiplexed format. Four different sizes of Qdots, with emissions at 525, 545, 565, and 585 nm are mixed with a polymer and coated onto the 8-mum-diameter magnetic microbeads to generate a nanobarcoded bead termed as QBeads. Twelve intensity levels for each of the four colors were used. Gene-specific oligonucleotide probes are conjugated to the surface of each spectrally nanobarcoded bead to create a multiplexed panel, and biotinylated cRNAs are generated from sample total RNA and hybridized to the gene probes on the microbeads. A fifth streptavidin Qdot (655 nm or infrared Qdot) binds to biotin on the cRNA, acting as a quantification reporter. Target identity was decoded based on spectral profile and intensity ratios of the four coding Qdots (525, 545, 565, and 585 nm). The intensity of the 655 nm Qdot reflects the level of biotinylated cRNA captured on the beads and provides the quantification for the corresponding target gene. The system shows a sensitivity of < or =10(4) target molecules detectable with T7 amplification, a level that is better than the 10(5) number achievable with a high-density microarray system, and approaching the 10(3)-10(4) level usually observed for quantitative PCR (qPCR). The QBead nanobarcode system has a dynamic range of 3.5 logs, better than the 2-3 logs observed on various microarray platforms. The hybridization reaction is performed in liquid phase and completed in 1-2 hours, at least 1 order of magnitude faster than microarray-based hybridizations. Detectable fold change is lower than 1.4-fold, showing high precision even at close to single copy per cell level. Reproducibility for this proof-of-concept study approaches that of Affymetrix GeneChip microarray, with an R(2) value between two repeats at 0.984, and interwell CV around 5%. In addition, it provides increased flexibility, convenience, and cost-effectiveness in comparison to conventional gene expression profiling methods.

Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater than 2-fold. Genes activated in treated cells included those involved in carbohydrate binding, intracellular vesicle formation, and cellular response to stress. Conversely, PEG-silane-Qdots induce a down-regulation of genes involved in controlling the M-phase progression of mitosis, spindle formation, and cytokinesis. Promoter analysis of these results reveals that expression changes may be attributed to the down-regulation of FOXM and BHLB2 transcription factors. Remarkably, PEG-silane-Qdots, unlike carbon nanotubes, do not activate genes indicative of a strong immune and inflammatory response or heavy-metal-related toxicity. The experimental evidence shows that CdSe/ZnS Qdots, if appropriately protected, induce negligible toxicity to the model cell system studied here, even when exposed to high dosages. This study indicates that PEG-coated silanized Qdots pose minimal impact to cells and are a very promising alternative to uncoated Qdots.

Molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast.

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Although both fullerene and carbon nanotubes have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types and carbon nanomaterials have been exploited for cancer therapies, the molecular and cellular mechanisms for cytotoxicity of this class of nanomaterial are not yet fully apparent. To address this question, we have performed whole genome expression array analysis and high content image analysis based phenotypic measurements on human skin fibroblast cell populations exposed to multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs). Here we demonstrate that exposing cells to MWCNOs and MWCNTs at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis. Expression array analysis indicates that multiple cellular pathways are perturbed after exposure to these nanomaterials at these doses, with material-specific toxigenomic profiles observed. Moreover, there are also distinct qualitative and quantitative differences in gene expression profiles, with each material at different dosage levels (6 and 0.6 microg/mL for MWCNO and 0.6 and 0.06 microg/mL for MWCNT). MWCNO and MWCNT exposure activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. MWCNTs induce genes indicative of a strong immune and inflammatory response within skin fibroblasts, while MWCNO changes are concentrated in genes induced in response to external stimuli. Promoter analysis of the microarray results demonstrate that interferon and p38/ERK-MAPK cascades are critical pathway components in the induced signal transduction contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs.