Understanding neurite growth regulation remains a seminal problem in neurobiology. During development and regeneration, neurite growth is modulated by neurotrophin-activated signaling endosomes that transmit regulatory signals between soma and growth cones. After injury, delivering neurotrophic therapeutics to injured neurons is limited by our understanding of how signaling endosome localization in the growth cone affects neurite growth. Nanobiotechnology is providing new tools to answer previously inaccessible questions. Here, we show superparamagnetic nanoparticles (MNPs) functionalized with TrkB agonist antibodies are endocytosed into signaling endosomes by primary neurons that activate TrkB-dependent signaling, gene expression and promote neurite growth. These MNP signaling endosomes are trafficked into nascent and existing neurites and transported between somas and growth cones in vitro and in vivo. Manipulating MNP-signaling endosomes by a focal magnetic field alters growth cone motility and halts neurite growth in both peripheral and central nervous system neurons, demonstrating signaling endosome localization in the growth cone regulates motility and neurite growth. These data suggest functionalized MNPs may be used as a platform to study subcellular organelle localization and to deliver nanotherapeutics to treat injury or disease in the central nervous system. axon | nanotechnology C entral nervous system (CNS) neurons fail to regenerate after injury or disease because of reduced intrinsic axon growth ability (1, 2), inhibitory molecules (3-6), and deficient neurotrophic factor signaling (7-9). Neurotrophins, like brain-derived neurotrophic factor (BDNF), activate tropomyosin-related kinase B (TrkB) receptors and are endocytosed by clathrin-dependent and -independent mechanisms into signaling endosomes (10, 11). These signaling endosomes signal persistently during retrograde (12) and anterograde (13, 14) transport in axons or dendrites (15, 16) directing neurite growth, survival, and cell migration (17, 18). Signaling endosomes are critical long-range communication links used by neurons in the central and peripheral nervous system during development and regeneration (17), whose dysfunction is linked to nervous system disorders (19-21). Therefore, studying signaling endosome localization and related functions in regulating neurite growth is vital. MNPs are emerging as flexible, multimodal nanoparticles that can be targeted to specific tissues or cells by molecular functionalization. To alter signaling endosome localization, we targeted functionalized MNPs to active TrkB signaling endosomes and demonstrate that magnetically manipulating their localization affects growth cone behavior and neurite growth. ResultsTo load MNPs into TrkB signaling endosomes, 50-nm MNPs were functionalized with the anti-TrkB agonist antibody, 29D7, conjugated to Alexa 594. 29D7 activates TrkB and enhances retinal ganglion cell (RGC) survival and neurite growth in vitro and in vivo (22). Now, we show 29D7 facilitates rapid MNP e...

PurposeMagnetic nanoparticles (MNPs) may be used for focal delivery of plasmids, drugs, cells, and other applications. Here we ask whether such particles are toxic to ocular structures.MethodsTo evaluate the ocular toxicity of MNPs, we asked if either 50 nm or 4 µm magnetic particles affect intraocular pressure, corneal endothelial cell count, retinal morphology including both cell counts and glial activation, or photoreceptor function at different time points after injection. Sprague-Dawley rats (n = 44) were injected in the left eye with either 50 nm (3 µl, 1.65 mg) or 4 µm (3 µl, 1.69 mg) magnetic particles, and an equal volume of PBS into the right eye. Electroretinograms (ERG) were used to determine if MNPs induce functional changes to the photoreceptor layers. Enucleated eyes were sectioned for histology and immunofluorescence.ResultsCompared to control-injected eyes, MNPs did not alter IOP measurements. ERG amplitudes for a-waves were in the 100–250 µV range and b-waves were in the 500–600 µV range, with no significant differences between injected and non-injected eyes. Histological sectioning and immunofluorescence staining showed little difference in MNP-injected animals compared to control eyes. In contrast, at 1 week, corneal endothelial cell numbers were significantly lower in the 4 µm magnetic particle-injected eyes compared to either 50 nm MNP- or PBS-injected eyes. Furthermore, iron deposition was detected after 4 µm magnetic particle but not 50 nm MNP injection.ConclusionsIntravitreal or anterior chamber injections of MNPs showed little to no signs of toxicity on retinal structure, photoreceptor function or aqueous drainage in the eye. Our results suggest that MNPs are safe for intraocular use.

Background: Magnetic nanoparticles may be used for focal delivery for cells, plasmids or drugs, and other applications. Here we asked whether magnetic nanoparticles could be detected in vivo at different time points after intravitreal injection by magnetic resonance imaging.

Perhaps the most exciting new platform for translating advances in the basic sciences to therapeutics for eye disease is in nanotechnology. Taking advantage of properties specific to particles on the nanometer scale, scientists are incorporating nanotechnology into new approaches for drug delivery, gene therapy and cell and tissue engineering. Applications for cornea, uveitis, glaucoma and retinal repair are fast approaching clinical use.

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein–protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches.