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Timothy E Kennedy

Publications and source records attributed to Timothy E Kennedy.

At least 19 recordsLinked to original sources
PubMed2007

Simvastatin regulates oligodendroglial process dynamics and survival.

Simvastatin, a lipophilic statin that crosses the blood-brain barrier, is being evaluated as a potential therapy for multiple sclerosis (MS) due to its anti-inflammatory properties. We assessed the effects of simvastatin on cultures of rat newborn and human fetal oligodendrocyte progenitor cells (OPCs) and human adult mature oligodendrocytes (OLGs) with respect to cellular events pertaining to myelin maintenance and repair. Short-term simvastatin treatment of OPCs (1 day) induced robust process extension, enhanced differentiation to a mature phenotype, and decreased spontaneous migration. These effects were reversed by isoprenoid products and mimicked with an inhibitor of Rho kinase (ROCK), the downstream effector of the isoprenylated protein RhoA GTPase. Prolonged treatment (2 days) caused process retraction that was rescued by cholesterol, and increased cell death (4 days) partially rescued by either cholesterol or isoprenoid co-treatment. In comparison, simvastatin treatment of human mature OLGs required a longer initial time course (2 days) to induce significant process outgrowth, mimicked by inhibiting ROCK. Prolonged treatment of mature OLGs was associated with process retraction (6 days) and increased cell death (8 days). Human-derived OPCs and mature OLGs demonstrated an increased sensitivity to simvastatin relative to the rodent cells, responding to nanomolar versus micromolar concentrations. Our findings indicate the importance of considering the short- and long-term effects of systemic immunomodulatory therapies on neural cells affected by the MS disease process. (c) 2006 Wiley-Liss, Inc.

Animals↗
PubMed2006

When a diffusible axon guidance cue stops diffusing: roles for netrins in adhesion and morphogenesis.

Netrins are a small family of secreted proteins that are best known for their role as secreted long-range chemotropic guidance cues. Extracellular gradients of netrin protein, established by diffusion, are thought to direct cell and axon migration during neural development. In addition to this long-range role, recent findings provide increasing support for short-range functions, in which secreted netrin protein remains closely associated with its cellular source. Emerging evidence for short-range actions of netrins suggests that they contribute to tissue morphogenesis by regulating cell-cell and cell-matrix adhesion.

Animals↗
PubMed2006

Axon guidance by diffusible chemoattractants: a gradient of netrin protein in the developing spinal cord.

Gradients of diffusible long-range attractant and repellent proteins have been proposed to guide growing axons during nervous system development, but such gradients have never been visualized directly. In the embryonic spinal cord, commissural axons pioneer a circumferential trajectory to the floor plate at the ventral midline directed by secreted proteins of the netrin family. In the embryonic chick spinal cord netrin-1 mRNA is expressed by floor plate cells and netrin-2 mRNA by neural epithelial cells. Antibodies to the two netrins reveal a gradient of netrin protein directly in the path of commissural axons. The netrin-1 gradient itself extends many cell diameters dorsal to the floor plate, the site of netrin-1 expression. A similar distribution of netrin-1 protein has been detected in embryonic rat and mouse spinal cord. The detection of a gradient of netrin-1 protein supports the operation of long-range chemotropic mechanisms in the developing nervous system.

Animals↗
PubMed2006

Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta.

The leukocyte common antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs), LAR, RPTP-sigma, and RPTP-delta, regulate neuroendocrine development, axonal regeneration, and hippocampal long-term potentiation in mammals. In Drosophila, RPTPs are required for appropriate axon targeting during embryonic development. In contrast, deletion of any one of the three LAR-RPTP family members in mammals does not result in gross axon targeting defects. Both RPTP-sigma and RPTP-delta are highly expressed in the developing mammalian nervous system, suggesting they might be functionally redundant. To test this hypothesis, we generated RPTP-sigma and RPTP-delta (RPTP-sigma/delta) double-mutant mice. Although embryonic day 18.5 RPTP-sigma and RPTP-delta single-mutant embryos were viable, RPTP-sigma/delta double mutants were paralyzed, were never observed to draw a breath, and died shortly after cesarean section. RPTP-sigma/delta double mutants exhibit severe muscle dysgenesis and severe loss of motoneurons in the spinal cord. Detailed analysis of the projections of phrenic nerves in RPTP-sigma/delta double mutants indicated that these motoneuron axons emerge normally from the cervical spinal cord, but stall on reaching the diaphragm. Our results demonstrate that RPTP-sigma and RPTP-delta complement each other functionally during mammalian development, and reveal an essential contribution of RPTP-sigma and RPTP-delta to appropriate motoneuron axon targeting during mammalian axonogenesis.

Animals↗
PubMed2006

Protein kinase A regulates the sensitivity of spinal commissural axon turning to netrin-1 but does not switch between chemoattraction and chemorepulsion.

Bifunctional axon guidance cues have been grouped into two classes depending on whether changes in intracellular cAMP or cGMP switch the response of the growth cone between attraction and repulsion. According to this model, axons respond to netrin-1, a group I guidance cue, as a chemoattractant when cAMP levels are high in the growth cone but switch and are repelled when the intraneuronal concentration of cAMP is low. The model is complicated by the proposal that cAMP-dependent kinase, protein kinase A (PKA), functions as a downstream effector for several guidance cues, including netrin-1, suggesting a close inter-relationship between guidance cue signal transduction and mechanisms regulating the switch between attraction and repulsion. Here, we examine possible interactions between netrin-1-mediated axon guidance and cAMP signaling in embryonic rat spinal commissural neurons. We report that netrin-1 does not alter the concentration of cAMP or PKA activity in these neurons across a wide range of netrin-1 concentrations and time points after application, leading us to conclude that netrin-1 does not regulate PKA in these cells. In contrast to the cyclic nucleotide switch model, we report that, despite inhibiting PKA, embryonic spinal commissural axons were always attracted to netrin-1 and never repelled. Instead, manipulating PKA regulated the sensitivity of chemoattraction to netrin-1: PKA inhibition reduced, and PKA activation increased, the distance over which axons turn toward a source of netrin-1. These findings indicate that the mechanisms underlying cyclic nucleotide-regulated switching are separable from the signal transduction mechanisms required for chemoattraction to netrin-1.

Animals↗
PubMed2006

Positioned to inhibit: netrin-1 and netrin receptor expression after spinal cord injury.

Netrin-1 regulates axon extension during embryonic development and is expressed by neurons and myelinating oligodendrocytes in the adult CNS. To investigate the potential role of netrin-1 after spinal cord injury, we examined the expression of netrin-1 and netrin receptors after sagittal myelotomy in adult rats. This lesion targets spinal commissural projections, which respond to netrin-1 during development. Netrin-1 mRNA and protein levels were dramatically reduced at the site of injury and reduced expression persisted for at least 7 months. Neither netrin-1 protein nor mRNA was associated with the glial scar, but netrin-1 was expressed by neurons and oligodendrocytes immediately adjacent to the lesion. The post-injury distribution detected is similar to that reported for myelin-associated inhibitors of axon regeneration, such as Nogo, and is distinct from the distribution of inhibitors associated with a glial scar. DCC and UNC-5 homologue (UNC5H) expression also was reduced after injury. Although UNC5H levels recovered, DCC expression at the site of injury remained approximately 50% of pre-injury values at 7 months. Increased UNC5H immunoreactivity was associated with fibers in the superficial layers of the dorsal horn and in fibers located in white matter adjacent to the lesion. The dominant expression of UNC5H on axons and neurons in the spinal cord after injury and the persistent expression of netrin-1 by oligodendrocytes surrounding the lesion are consistent with the hypothesis that netrin-1 is a myelin-associated inhibitor of axonal regeneration after spinal cord injury.

Animals↗
PubMed2005

Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord.

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Accessory Nerve↗
PubMed2005

Deleted in colorectal cancer binding netrin-1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N-WASP into an intracellular signaling complex that promotes growth cone expansion.

Extracellular cues direct axon extension by regulating growth cone morphology. The netrin-1 receptor deleted in colorectal cancer (DCC) is required for commissural axon extension to the floor plate in the embryonic spinal cord. Here we demonstrate that challenging embryonic rat spinal commissural neurons with netrin-1, either in solution or as a substrate, causes DCC-dependent increases in growth cone surface area and filopodia number, which we term growth cone expansion. We provide evidence that DCC influences growth cone morphology by at least two mechanisms. First, DCC mediates an adhesive interaction with substrate-bound netrin-1. Second, netrin-1 binding to DCC recruits an intracellular signaling complex that directs the organization of actin. We show that netrin-1-induced growth cone expansion requires Cdc42 (cell division cycle 42), Rac1 (Ras-related C3 botulinum toxin substrate 1), Pak1 (p21-activated kinase), and N-WASP (neuronal Wiskott-Aldrich syndrome protein) and that the application of netrin-1 rapidly activates Cdc42, Rac1, and Pak1. Furthermore, netrin-1 recruits Cdc42, Rac1, Pak1, and N-WASP into a complex with the intracellular domain of DCC and Nck1. These findings suggest that DCC influences growth cone morphology by acting both as a transmembrane bridge that links extracellular netrin-1 to the actin cytoskeleton and as the core of a protein complex that directs the organization of actin.

Analysis of Variance↗
PubMed2005

Receptor protein tyrosine phosphatase sigma inhibits axon regrowth in the adult injured CNS.

Recently, receptor protein tyrosine phosphatase-sigma (RPTPsigma) has been shown to inhibit axon regeneration in injured peripheral nerves. Unlike the peripheral nervous system (PNS), central nervous system (CNS) neurons fail to regenerate their axons after injury or in disease. In order to assess the role of RPTPsigma in CNS regeneration, we used the retinocollicular system of adult mice lacking RPTPsigma to evaluate retinal ganglion cell (RGC) axon regrowth after optic nerve lesion. Quantitative analysis demonstrated a significant increase in the number of RGC axons that crossed the glial scar and extended distally in optic nerves from RPTPsigma (-/-) mice compared to wild-type littermate controls. Although we found that RPTPsigma is expressed by adult RGCs in wild-type mice, the retinas and optic nerves of adult RPTPsigma (-/-) mice showed no histological defects. Furthermore, the time-course of RGC death after nerve lesion was not different between knockout and wild-type animals. Thus, enhanced axon regrowth in the absence of RPTPsigma could not be attributed to developmental defects or increased neuronal survival. Finally, we show constitutively elevated activity of mitogen-activated protein kinase (MAPK) and Akt kinase in adult RPTPsigma (-/-) mice retinas, suggesting that these signaling pathways may contribute to promoting RGC axon regrowth following traumatic nerve injury. Our results support a model in which RPTPsigma inhibits axon regeneration in the adult injured CNS.

Animals↗
PubMed2005

Membrane-associated guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived serotonergic axons.

Unlike many neurons that extend an axon precisely to a single target, individual dorsal raphe 5-HT neurons project to multiple brain regions and their axon terminals often lack classical synaptic specializations. It is not known how 5-HT axon collaterals select between multiple target fields, or even if 5-HT axons require specific guidance cues to innervate their targets. Nor is it known how these axon collaterals are restrained within specific innervation target regions. To investigate this, we challenged explants of dorsal raphe with co-explants, or cell membrane preparations of ventral midbrain, striatum or cerebral cortex. We provide evidence for membrane-associated cues that promote 5-HT axon growth into each of these three target regions. The axon growth-promoting activity was heat-, protease- and phosphatidylinositol-phospholipase-C (PI-PLC)-sensitive. Interestingly, 5-HT axons specifically lost the ability to grow in heterotypic explants, or membrane carpets, following contact with ventral midbrain or striatal, but not cortical, explants or membranes. This inductive activity associated with striatal and ventral midbrain membranes was sensitive to both high salt extraction and PI-PLC treatment. By contrast, the activity that inhibited 5-HT axon growth onto heterotypic membranes was sensitive only to high salt extraction. These results provide evidence that a glycosylphosphatidylinositol (GPI)-linked membrane protein promotes 5-HT axon growth, and that short-range membrane-bound, as well as GPI-linked, molecules contribute to the guidance of 5-HT axon collaterals. These findings suggest that 5-HT axon collaterals acquire a target-induced growth-inhibitory response to alternative targets, increasing their selectivity for the newly innervated field.

Age Factors↗
PubMed2004

Developmental shift in expression of netrin receptors in the rat spinal cord: predominance of UNC-5 homologues in adulthood.

Netrins are a family of secreted proteins required for normal neural development. Netrin-1 is expressed at similar levels in the adult rat spinal cord and the embryonic CNS, suggesting that it contributes to adult CNS function. Here we show that the netrin receptors dcc, neogenin, unc5h1, unc5h2, and unc5h3 are also expressed in the adult rat spinal cord. Lower levels of DCC and neogenin were detected in the adult relative to the embryonic CNS. Conversely, the adult spinal cord contains increased levels of UNC-5 homologues in comparison with the embryo. Multiple mRNA transcripts detected by Northern blot analysis suggested that netrin receptors might be encoded by alternatively spliced mRNAs. We have identified a novel alternatively spliced mRNA encoding UNC5H1, UNC5H1(Delta)TSP1, which lacks the first of the two extracellular thrombospondin domains. This novel splice variant is the major transcript detected in the early embryonic CNS, although both splice variants are expressed in the adult. Previously identified alternatively spliced mRNAs encoding DCC and neogenin were also detected. Dcc, neogenin, unc5h1, unc5h2, and unc5h3 are expressed by subsets of neurons. Robust expression of unc5h2 was found in glia. These findings suggest that unc-5 homologues constitute a major mode of netrin-1 signal transduction in the adult spinal cord and may be involved in phenomena analogous to axon repulsion, such as inhibiting process extension and collateral sprouting.

Age Factors↗
PubMed2004

Protein kinase A activation promotes plasma membrane insertion of DCC from an intracellular pool: A novel mechanism regulating commissural axon extension.

Protein kinase A (PKA) exerts a profound influence on axon extension during development and regeneration; however, the molecular mechanisms underlying these effects of PKA are not understood. Here, we show that DCC (deleted in colorectal cancer), a receptor for the axon guidance cue netrin-1, is distributed both at the plasma membrane and in a pre-existing intracellular vesicular pool in embryonic rat spinal commissural neurons. We hypothesized that the intracellular pool of DCC could be mobilized to the plasma membrane and enhance the response to netrin-1. Consistent with this, we show that application of netrin-1 causes a modest increase in cell surface DCC, without increasing the intracellular concentration of cAMP or activating PKA. Intriguingly, activation of PKA enhances the effect of netrin-1 on DCC mobilization and increases axon extension in response to netrin-1. PKA-dependent mobilization of DCC to the plasma membrane is selective, because the distributions of transient axonal glycoprotein-1, neural cell adhesion molecule, and trkB are not altered by PKA in these cells. Inhibiting adenylate cyclase, PKA, or exocytosis blocks DCC translocation on PKA activation. These findings indicate that netrin-1 increases the amount of cell surface DCC, that PKA potentiates the mobilization of DCC to the neuronal plasma membrane from an intracellular vesicular store, and that translocation of DCC to the cell surface increases axon outgrowth in response to netrin-1.

Adenylyl Cyclase Inhibitors↗
PubMed2004

Oligodendrocyte precursors on the move: mechanisms directing migration.

Oligodendrocyte precursor cells traverse long distances to reach their axonal targets. The molecules that influence their migration include a combination of short-range attractants and repellents and long-range chemoattractants and chemorepellents. Here, the authors review mechanisms that direct oligodendrocyte precursor cells as they migrate throughout the developing CNS.

Animals↗
PubMed2003

Netrin-1 is a chemorepellent for oligodendrocyte precursor cells in the embryonic spinal cord.

Netrin-1, secreted by floor plate cells, orients axon extension in relation to the ventral midline of the embryonic spinal cord. Oligodendrocyte precursor (OP) cells are born close to the ventral midline and migrate away from the floor plate. Here we show that OP cells, identified by expression of the platelet-derived growth factor alpha receptor, express the netrin receptors dcc and unc5h1 but do not express netrin-1. Using a microchemotaxis assay, we demonstrate that migrating OPs are repelled by a gradient of netrin-1 in vitro. Furthermore, application of netrin-1 to OPs in vitro triggers retraction of OP processes. In the absence of netrin-1 or Deleted in Colorectal Cancer (DCC) function in vivo, fewer OP cells migrate from the ventral to the dorsal embryonic spinal cord, consistent with netrin-1 acting as a repellent. In addition to their role regulating cell movement, DCC and UNC-5 homologs have been suggested to function as proapoptotic dependence receptors, triggering cell death in the absence of netrin-1. In contrast, we report no evidence of increased OP cell death in vivo or in vitro in the absence of either netrin-1 or DCC. These findings indicate that netrin-1 is a repellent cue for migrating OPs in the embryonic spinal cord.

Animals↗
PubMed2003

Glutamate promotes proliferation of striatal neuronal progenitors by an NMDA receptor-mediated mechanism.

Increasing evidence suggests that classical neurotransmitters play important roles in the development of the mammalian CNS. We used in vivo and in vitro models to identify a novel role for glutamate in striatal neurogenesis mediated by NMDA receptors. In utero exposure to NMDA receptor antagonists during striatal neurogenesis caused a dramatic reduction in the total number of adult striatal neurons. In contrast, embryos exposed to NMDA receptor antagonists immediately after the main period of neurogenesis showed no significant change in neuronal number in the adult striatum. In addition, examination of embryos shortly after NMDA receptor blockade revealed reduced proliferation in the lateral ganglionic eminence (LGE). In culture, dividing neuronal progenitors derived from the embryonic LGE showed marked reduction in 5'-bromodeoxyuridine (BrdU) uptake when exposed to NMDA receptor antagonists, indicating reduced DNA synthesis. Low concentrations of NMDA significantly increased proliferation, whereas high concentrations were toxic. AMPA-KA receptor antagonists had no significant effect on striatal neuroblast proliferation either in vivo or in vitro. These results support the hypothesis that glutamate plays a novel role during early development of the ventral telencephalon, promoting proliferation of striatal neuronal progenitors by an NMDA receptor-dependent mechanism. In contrast, previous findings suggest that proliferation of cortical progenitors derived from the dorsal telencephalon is regulated by activation of AMPA-KA but not NMDA receptors. Heterogeneous responses to glutamate in different germinal zones of the telencephalon may be an important mechanism contributing to generating neuronal diversity in the forebrain.

Animals↗
PubMed2003

Visualization of the dendritic arbor of neurons in intact 500 microm thick brain slices.

Characterizing the structure and electrophysiological properties of single neurons is essential for understanding how individual cells contribute to the function of neuronal networks. Following intra-cellular recording from neurons in acute brain slices, the structure of the recorded cell has typically been examined by serial sectioning of the tissue slice and then reconstructing the neuron of interest; a labor-intensive and time-consuming process. Here, we have adapted a whole-mount immunohistochemical technique and used it to visualize the dendritic arbor of individual neurons in sections of adult CNS tissue up to 500 microm thick. Permeabilization of the slice and extensive washing allow histochemical reagents to penetrate and be washed from the section, producing limited background staining. Using this method, the cell within the slice can be sectioned optically and reconstructed using the optical sections. We present images of the dendritic trees of neurons in 500 microm thick slices of adult rat entorhinal cortex and hippocampus, labeled either immunohistochemically, or by biocytin injection following whole-cell patch clamp or sharp electrode recordings. The resolution obtained is sufficient to visualize dendritic spines deep within the section. The method is free from artifacts associated with cutting serial sections and is broadly applicable to tasks that require visualization of the fine structure of individual cells in thick slices of CNS tissue.

Animals↗
PubMed2002

Crossing the ventral midline causes neurons to change their response to floor plate and alar plate attractive cues during transmedian migration.

Neuronal migration is required for the establishment of specific neural structures, such as layers and nuclei. Neurons migrate along specific migratory routes toward their final destinations, sometimes across long distances. However, the cellular and molecular interactions that control neuronal migration are largely unknown. Here, we examined the mechanism underlying the transmedian migration of precerebellar neurons using a flat whole-mount preparation of the rat embryo. These neurons were initially attracted by the floor plate (FP) at the ventral midline. However, after crossing the midline, they lost their responsiveness to the FP and became attracted by the alar plate (AP). Although the loss of responsiveness to FP cues was caused by an encounter of migrating cells with the FP, the gain of responsiveness to AP cues occurred irrespective of their encounter with the FP. These results identify a crucial change in the response of migrating cells to attractive guidance cues during the transmedian migration of precerebellar neurons.

Animals↗