We tested the hypothesis that netrin is as an inhibitor to axon growth in adult white matter. An in vitro assay was established to neutralize potential netrin-mediated inhibition of axon outgrowth via Unc5 receptor signaling. Embryonic day 15 (E15) rat spinal cords were isolated, motor columns were dissected and Unc5H1- and Unc5H3- expressing motor neurons were enzymatically dissociated and plated on coverslips that were coated with myelin prepared from adult rat spinal cord. Unc5H2 receptor bodies, fusion proteins between the extracellular domain of the netrin receptor Unc5H2 and human Fc IgG, were engineered to neutralize netrin-1. Axon outgrowth from E15 spinal motor neurons increased by 82% (p < 0.001) after treatment of myelin with soluble Unc5H2 receptor bodies, from a mean neurite length of 49 µm +/- 0.6 µm (SEM) using control Fc to 89 µm +/- 1.3 µm (SEM) using the receptor body. Effects were observed in a dose dependent manner. These results suggest that physiological levels of netrin-1 contained w...

Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of...

Acute trauma to the spinal cord in adult mammals can result in permanent loss of function due to scant regeneration of injured axons. The injured axons encounter several barriers to regeneration, one of which is the astroglial scar at the injury site. The astroglial scar contains extracellular matrix (ECM) molecules deposited by reactive astrocytes in response to the injury. It has been shown that one such class of ECM molecules, the chondroitin sulfate proteoglycans (CSPGs), can impede axonal regeneration in vivo as well as inhibit neurite outgrowth in vitro. All CSPGs consist of carbohydrate glycosaminoglycan (GAG) chains attached to a core protein. Chondroitinase ABC, an enzyme that removes the GAG chains, has been shown to facilitate axon regrowth and improve functional recovery in a rat model of spinal cord injury (SCI). However, it has also been found that the core protein can retain inhibitory effects on axon outgrowth. Thus, degrading the core protein of CSPGs, in addition to removing their GAG cha...

A detailed knowledge of axon guidance mechanisms is essential to understanding neural development, providing insights into the molecular pathways associated with diseases of the nervous system. The visual system of Drosophila offers an amenable model in which to study axon guidance, as retinal (R-cell) axons project over long distances through a complex cellular milieu to synapse with appropriate targets in a precise topographic array. This precise architecture of axon connections is established using information encoded by phylogenetically conserved guidance molecules, although the complete repertoire and functional roles of these molecules are not known. We report here a role for the Semaphorin receptor, Plexin-B, in mediating R-cell axon target selection in the developing Drosophila visual system. Normal Plexin-B signaling was disrupted using the UAS-GAL4 system to drive temporal and tissue-specific transgene expression in developing optic ganglia. Visual system architecture was subsequently analyzed at...

Raf kinases mediate extracellular signaling via receptor tyrosine kinases. In vitro evidence has implicated Raf kinases in survival and morphological responses to neurotrophins. Because both B-Raf and Raf-1 (C-Raf) null mice die at mid-embryonic stages, the role of Raf signaling in nervous system development has not been defined. We constructed a B-Raf floxed allele with loxP sites flanking the N-terminus of the Ras binding domain. A second B-Raf floxed line, designed to allow truncation of the kinase domain was kindly provided by Alcino Silva (UCLA). Conditional ablation using a Nestin-Cre deleter showed that B-Raf protein levels were reduced by 90% in the nervous system of young adults. Furthermore, baseline p42/44-ERK phosphorylation in the brain, spinal cord and DRGs was dramatically reduced. B-Raf f/-:NesCre mice on a mixed Bl6/129 background exhibited a severe growth deficit from P15 onwards and exhibited modestly reduced brain size and cortical thinning. Surprisingly, DRG sizes were normal in B-Raf ...

Current CNS injury models for studying of remyelination are associated with substantial collateral tissue damage. Here we demonstrate synchrotron-generated, high dosage x-ray microplanar beams (microbeams) as a novel tool for producing targeted and selective demyelination. Spinal cords from rats were transaxially irradiated using 270 µM beams with incident doses ranging from 500-2000 Gy. At doses ranging from 1000-1250 Gy, immunohistochemical and electron microscope data revealed significant demyelination in white matter with minimal axon damage at three months following exposure. We also observed ablation of neurons in segmental gray matter in the microbeam path. Substantial cellular and tissue recovery occurred in white matter. In contrast, we observed an irreversible loss of axons and neurons in gray matter. In general, axonal demyelination was more prevalent in gray matter than white matter and amongst axons of small diameter than large diameter. We observed complete sparing of tissue immediately perip...

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on ...

Traumatic peripheral nerve injuries affect thousands of people every year. These injured nerves have the ability to regenerate, but the process is slow and full functional recovery is rare for most patients. Therapy in the form of treadmill exercise has...

The axon initial segment (AIS), a specific region for action potential initiation in neurons, is a critical determinant of neuronal excitability. Growing evidence indicates that the appropriate recruitment of the AIS macrocomplex is essential for synchr...

Peripheral axon regeneration is improved when the nerve lesion under consideration has recently been preceded by another nerve injury. This is known as the conditioning lesion effect (CLE). While the CLE is one of the most well robust and well character...