Helen M. Cooper

The Wnt Receptor Ryk Is Required for Wnt5a-Mediated Axon Guidance on the Contralateral Side of the Corpus Callosum

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk–Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, in Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk−/− mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk−/− embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

Subcellular compartment targeting of layered double hydroxide nanoparticles

Current investigations show that layered double hydroxide (LDH) nanoparticles have high potential as effective non-viral agents for cellular drug delivery due to their low cytotoxicity, good biocompatibility, high drug loading, control of particle size and shape, targeted delivery and drug release control. Two types of Mg(2)Al-LDH nanoparticles with fluorescein isothiocyanate (FITC) were controllably prepared. One is morphologically featured as typical hexagonal sheets (50-150 nm laterally wide and 10-20 nm thick), while the other as typical rods (30-60 nm wide and 100-200 nm long). These LDH(FTIC) nanoparticles are observed to immediately transfect into different mammalian cell lines. We found that internalized LDH(FITC) nanorods are quickly translocated into the nucleus while internalized LDH(FITC) nanosheets are retained in the cytoplasm. Inhibition experiments show that the cellular uptake is a clathrin-mediated time- and concentration-dependent endocytosis. Endosomal escape of LDH(FITC) nanoparticles is suggested to occur through the deacidification of LDH nanoparticles. Since quick nuclear targeting of LDH(FITC) nanorods requires an active process, and although the exact mechanism is yet to be fully understood, it probably involves an active transport via microtubule-mediated trafficking processes. Targeted addressing of two major subcellular compartments by simply controlling the particle morphology/size could find a number of applications in cellular biomedicine.

Efficient delivery of siRNA to cortical neurons using layered double hydroxide nanoparticles

Small interfering RNAs (siRNAs) are capable of targeting and destroying specific mRNAs, making them particularly suited to the treatment of neurodegenerative conditions such as Huntingtons Disease where the production of abnormal proteins results in a gain-of-function phenotype. Although a variety of nanoparticle formulations are currently under development as siRNA delivery systems, application of these technologies has been limited by their high cytotoxicity, low drug loading capacity and release, and inability to penetrate cell membranes. Layered double hydroxide (LDH) nanoparticles are now emerging as a potential new drug delivery system as they exhibit low cytotoxicity and are highly biocompatible. Here we present the first study investigating LDH delivery of siRNAs to primary cultured neurons. We show that internalization by neurons is rapid, dose-dependent and saturable, and markedly more efficient than in other cell types. We demonstrate that siRNA-LDH complexes are internalized by clathrin-dependent endocytosis at the cell body and in neurites, with subsequent retrograde transport to the cell body followed by efficient release into the cytoplasm. Finally we show that LDH mediated siRNA delivery effectively silences neuronal gene expression. This study therefore confirms the potential of LDH nanoparticles as a drug delivery system for patients suffering from neurodegenerative disease.

Layered double hydroxide nanoparticles as cellular delivery vectors of supercoiled plasmid DNA

Within the family of nanomaterials, carbon nanotubes (CNTs) have emerged as a new efficient scaffold for studying molecular interactions at interfaces. Poor dispersability of CNTs in any solvent presents a considerable drawback for the development of novel functional composite structures. Previous studies have demonstrated that the solubility of CNTs can be greatly enhanced by employing appropriate surfactants, some of them being biological molecules. In this work, we study the noncovalent wrapping of lipid chains onto the graphitic surface of single-walled material (SWCNTs) by electron microscopy and Raman spectroscopy. Stable and homogenous aqueous suspensions of SWCNTs in the presence of lipids have been prepared, whereas their electrophoretic mobility was confirmed by ζ-potential measurements. Raman measurements revealed that smaller diameter SWCNTs are preferentially dispersed by lipid molecules in the aqueous supernatant part of the prepared suspension.We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the presence of LDH nanoparticles at different concentrations was investigated. This revealed 50% cell viability at 500 μg/mL of LDH nanoparticles that is much higher than 50–100 μg/mL used for the delivery tests. The supercoiled pEF-eGFP plasmid (ca. 6100 base pairs) was mixed with LDH nanoparticle suspensions for anion exchange at a weight ratio of DNA/LDH between 1:25 and 1:100. In vitro experiments show that GFP expression in HEK 293T cells starts in the first day, reaches the maximum levels by the second day and continues in the third day. The GFP expression generally increases with the increase in DNA loading in DNA-LDH nanohybrids. However, the delivery efficiency with LDH nanoparticles as the agent is low. For example, the relative efficiency is 7%–15% of that of the commercial agent FuGENE®6. Three to 6% of total cells expressed GFP in an amount detectable by the FACS cytometry 2 days after transfection at 1 μg/mL of plasmid DNA with 25 μg/mL of LDH nanomaterial. The lower delivery efficiency could be attributed to the aggregation of LDH nanoparticles caused by the long-chain plasmid DNA.To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (αCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

The human Sirt3 protein deacetylase is exclusively mitochondrial.

It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while SIRT3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established. Although mouse SIRT3 was recently shown to be mitochondrial, HsSIRT3 (human SIRT3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. In the present study we show, using various HsSIRT3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation, that in contrast with earlier reports HsSIRT3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition we suggest, on the basis of the analysis of public genome databases, that the full-length mouse SIRT3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.

Expression of the Netrin‐1 receptor, deleted in colorectal cancer (DCC), is largely confined to projecting neurons in the developing forebrain

Axon guidance mechanisms are crucial to the development of an integrated nervous system. One family of molecules that may be important in establishing axonal connectivity in mammals is the Netrins, and their putative receptors DCC (deleted in colorectal cancer), Neogenin, and Unc‐5. Knockout and mutational analyses of some of these genes have shown that they are critically involved in the development of several specific pathways in the developing brain. However, previous expression analyses of these genes have largely been confined to the developing spinal cord. In the present study, we analyzed the expression of DCC in the developing mouse forebrain. We found that DCC protein is expressed in specific axonal populations projecting from the developing olfactory bulb, neocortex, hippocampus, and epithalamus/habenular complex. In the developing olfactory bulb and neocortex, DCC expression is particularly evident during the targeting phase of axon outgrowth and is then rapidly downregulated. As predicted from the knockout and mutational analyses of this gene, DCC is expressed in axonal commissures, in particular the corpus callosum, hippocampal commissure, and the anterior commissure. In addition, we found that DCC is expressed in the habenular commissure, the fasciculus retroflexus, and the stria medularis. Therefore, this analysis implicates a function for DCC in additional axonal guidance systems not predicted from the knockout and mutational analyses. J. Comp. Neurol. 416:201–212, 2000.

Netrin-1 : Diversity in development

In 1990, the discovery of three Caenorhabditis elegans genes (unc5, unc6, unc40) involved in pioneer axon guidance and cell migration marked a significant advancement in neuroscience research [Hedgecock EM, Culotti JG, Hall DH. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 1990;4:61-85]. The importance of this molecular guidance system was exemplified in 1994, when the vertebrate orthologue of Unc6, Netrin-1, was discovered to be a key guidance cue for commissural axons projecting toward the ventral midline in the rodent embryonic spinal cord [Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 1994;78:409-424]. Since then, Netrin-1 has been found to be a critical component of embryonic development with functions in axon guidance, cell migration, morphogenesis and angiogenesis. Netrin-1 also plays a role in the adult brain, suggesting that manipulating netrin signals may have novel therapeutic applications.

Emerging roles for neogenin and its ligands in CNS development

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin‐1–neogenin interactions mediate chemoattractive axon guidance, while RGMa–neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin‐1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.

Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors

Background Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors. Principal Findings Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex. Conclusions Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

The Netrin Receptor DCC Is Required in the Pubertal Organization of Mesocortical Dopamine Circuitry

Netrins are guidance cues involved in neural connectivity. We have shown that the netrin-1 receptor DCC (deleted in colorectal cancer) is involved in the functional organization of the mesocorticolimbic dopamine (DA) system. Adult mice with a heterozygous loss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty, a critical period in the maturation of the mesocortical DA projection. Here, we examined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuit at puberty. Immunolabeling experiments in wild-type mice demonstrated that DCC is segregated to TH-positive fibers innervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty.