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Biomedical subjects

W D Snider

Publications and source records attributed to W D Snider.

At least 19 recordsLinked to original sources
PubMed2001

Different signaling pathways mediate regenerative versus developmental sensory axon growth.

Recent advances in defining neurotrophin signaling mediators have provided insights into the signal transduction mechanisms that underlie axon growth. Evidence is accumulating that major Trk effectors regulate the morphological development of embryonic peripheral neurons. Less is known about signaling related to the robust axon extension that follows peripheral axotomy of adult neurons. Regenerative axon growth can be mimicked in vitro by a "conditioning" lesion performed 2 weeks before culture (Smith and Skene, 1997). Previous work has implicated both neurotrophins and cytokines in this response. Because signal transduction mediators of both of these families of growth factors are well characterized, we have compared the role of neurotrophin and cytokine signaling in developmental versus regenerative sensory axon growth. Chemical inhibitors were administrated to embryonic and axotomized sensory neurons in vitro to block the activation of Erk kinase (MEK)-extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI3-K), and janus kinase (JAK) signaling. As expected, both MEK and PI3-K inhibition blocked axon growth from both naive and NGF-stimulated embryonic day 13 sensory neurons, whereas inhibition of JAK phosphorylation had no effect. In contrast, neither MEK nor PI3-K inhibitors blocked elongation of adult sensory neurons after a conditioning lesion. However, the addition of a JAK2 inhibitor prevented the regenerative axon response. Consistent with these pharmacological results, the percentage of neurons showing intense nuclear signal transducers and activators of transcription 3 phosphorylation after a conditioning lesion was markedly increased compared with controls. These observations demonstrate that the signaling mediators that underlie regenerative axon growth are distinct from those used during development and suggest that cytokine signaling may be critical to peripheral nervous system regeneration.

Animals↗
PubMed2001

Glial cell line-derived neurotrophic factor administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) in embryonic muscle fibers causes dramatic hyperinnervation of neuromuscular junctions. However, it is not known whether GDNF induces the extra innervation by regulation of axonal branching and/or synaptic maintenance. To address this issue, high levels of circulating GDNF were established by administering subcutaneous injections starting either at birth or later and continuing for up to 40 d. Treatment with exogenous GDNF beginning in the first week, but not later, increased the number of axons converging at neuromuscular junctions. The effect of GDNF on the branching pattern of individual motor axons was determined by reconstructing labeled axonal arbors from transgenic mice expressing yellow fluorescent protein in subsets of motor neurons. Whereas, at postnatal day 8 (P8) individual axons in control animals branched to sporadically innervate junctions within circumscribed regions of the muscle, motor units from GDNF injected animals had significantly more axonal branches and exhibited a high degree of localized arborization such that adjacent muscle fibers were often innervated by the same axon. Administration beginning at P0 and continuing through P40 prolonged multiple innervation of most fibers throughout the period of injection. Between P30 and P40 there was no net change in multiple innervation, although there was evidence of retraction bulbs, suggesting that axon extension and retraction were in equilibrium. We conclude that GDNF has a developmentally regulated effect on presynaptic branching and that sustained administration of GDNF induces a state of continuous synaptic remodeling.

Aging↗
PubMed2001

Neurotrophin receptor expression in retrogradely labeled trigeminal nociceptors--comparisons with spinal nociceptors.

In situ hybridization for trkA mRNA in trigeminal ganglion neurons retrogradely labeled with FluoroGold from the mandibular incisor demonstrated limited expression of the high-affinity nerve growth factor (NGF) receptor in this presumptive nociceptor population. Immunocytochemistry using polyclonal anti-trkA antibodies confirmed this result and extended it to show low levels of trkA protein expression in afferents labeled from the cornea. Less than 10% of the cells innervating the incisor, and approximately 15% of those innervating the cornea, were trkA-positive in adult and neonatal mice. This proportion is considerably lower than that observed in Dorsal Root Ganglion nociceptors, in which approximately 80% in neonates and approximately 40% in adults express trkA (Molliver and Snider, J. Comp Neurol 381: 428-438, 1997). Presumptive trigeminal nociceptors were further identified on the basis of expression of Calcitonin gene related peptide. In the entire ganglion, approximately 43% of the trkA-positive cells were CGRP-positive, and approximately 44% of the CGRP-positive cells were trkA-positive. Most trkA-positive cells that were CGRP-negative were medium-to-large diameter, while most of those that were CGRP-positive but trkA-negative were small diameter. Only approximately 5% of trkA-positive cells labeled from the incisor, and approximately 10% from the cornea, were CGRP-positive. Approximately 15% of the corneal or pulpal afferent neurons expressed ret-immunoreactivity. These results suggest that trigeminal nociceptors differ from spinal nociceptors in several significant ways. Differences in neurotrophic requirements may be related to differences in target tissues, in embryonic origin of some trigeminal ganglion cells, or in the timing of down-regulation of trkA expression in trigeminal ganglion cells.

Animals↗
PubMed2000

Glial cell line-derived neurotrophic factor and developing mammalian motoneurons: regulation of programmed cell death among motoneuron subtypes.

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF after in utero treatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability. In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRalpha1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRalpha2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRalpha1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRalpha1/GFRalpha2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRalpha2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Animals↗
PubMed2000

The transmembrane protein semaphorin 6A repels embryonic sympathetic axons.

Semaphorin 6A (Sema6A) (previously named Semaphorin VIa) is the originally described member of the vertebrate semaphorin class 6, a group of transmembrane semaphorins homologous to the insect semaphorin class 1. Although Sema-1a (previously named semaphorin I) has been implicated in axon guidance in insects, the function of Sema6A is currently unknown. We have expressed the extracellular domain of Sema6A in mammalian cells as either a monomeric or a dimeric fusion protein and tested for potential axon guidance effects on two populations of embryonic neurons in growth cone collapse and collagen matrix chemorepulsion assays. Sema6A was observed to induce growth cone collapse of sympathetic neurons with an EC50 of approximately 200 pM, although a 10-fold higher (EC50 of approximately 2 nM) concentration was necessary to induce growth cone collapse of dorsal root ganglion neurons. The activity of Sema6A is likely to depend on protein dimerization or oligomerization. Although Sema6A mRNA is expressed in complex patterns during embryonic development, it is strikingly absent from sympathetic ganglia. Sema6A is, however, expressed in areas avoided by sympathetic axons and in areas innervated by sympathetics, but before their arrival. Our results demonstrate that transmembrane semaphorins, like the secreted ones, can act as repulsive axon guidance cues. Our findings are consistent with a role for Sema6A in channeling sympathetic axons into the sympathetic chains and controlling the temporal sequence of sympathetic target innervation.

Animals↗
PubMed2000

Differential effects of TrkC isoforms on sensory axon outgrowth.

Neurotrophins are powerful regulators of neuronal morphology. Several lines of evidence are consistent with the idea that characteristic axonal and dendritic morphologies throughout the nervous system may be determined by local patterns of neurotrophin and neurotrophin receptor expression. Neurotrophin receptor tryosine kinases (Trks) exist in both tyrosine-containing (TK+) and tyrosine-lacking (TK-) isoforms, both of which are expressed in many neuronal populations. However, ratios of TK+ to TK- isoforms may vary at different stages of development and may be differentially distributed to cellular compartments. To test whether these isoforms have different functions related to axon outgrowth, full-length or tyrosine kinase-lacking TrkC receptors were overexpressed in embryonic dorsal root ganglion neurons maintained in explant cultures in neurotrophin-3 (NT-3)-containing media. Neurons were transfected with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) and TrkC receptor isoforms by particle-mediated gene transfer. Control neurons possessed 3.7 +/- 1.3 primary processes and 113.8 +/- 46 branch points. About 80% of the branches were located along the distal part of the axon. Transfection with the trkC TK+ increased the number of primary processes (6.5 +/- 2.8), whereas transfection with trkC TK- reduced the formation of primary processes (3.0 +/- 1.3). Surprisingly, the distribution of branch points was shifted to the proximal region of axons in neurons transfected with trkC TK-. These observations are consistent with the idea that differential expression of Trk isoforms during development may sculpt axonal morphology.

Animals↗
PubMed2000

Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration.

Evidence garnered from both human autopsy studies and genetic animal models has suggested a potential role for astrocytes in the pathogenesis of amyotrophic lateral sclerosis (ALS). Currently, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) represent the only known cause of motoneuron loss in the disease, producing 21q linked familial ALS (FALS). To determine whether astrocytic dysfunction has a primary role in familial ALS, we have generated multiple lines of transgenic mice expressing G86R mutant SOD1 restricted to astrocytes. In GFAP-m SOD1 mice, astrocytes exhibit significant hypertrophy and increased GFAP reactivity as the animals mature. However, GFAP-mutant SOD1 transgenic mice develop normally and do not experience spontaneous motor deficits with increasing age. Histological examination of spinal cord in aged GFAP-mSOD1 mice reveals normal motoneuron and microglial morphology. These results indicate that 21q linked FALS is not a primary disorder of astrocytes, and that expression of mutant SOD1 restricted to astrocytes is not sufficient to cause motoneuron degeneration in vivo. Expression of mutant SOD1 in other cell types, most likely neurons, is critical for the initiation of disease.

Aging↗
PubMed2000

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.

Animals↗
PubMed1999

Neurotrophins support the development of diverse sensory axon morphologies.

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax-/- neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.

Animals↗
PubMed1999

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

Animals↗
PubMed1998

Hyperinnervation of neuromuscular junctions caused by GDNF overexpression in muscle.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) by muscle greatly increased the number of motor axons innervating neuromuscular junctions in neonatal mice. The extent of hyperinnervation correlated with the amount of GDNF expressed in four transgenic lines. Overexpression of GDNF by glia and overexpression of neurotrophin-3 and neurotrophin-4 in muscle did not cause hyperinnervation. Thus, increased amounts of GDNF in postsynaptic target cells can regulate the number of innervating axons.

Animals↗
PubMed1998

Widespread elimination of naturally occurring neuronal death in Bax-deficient mice.

The proapoptotic molecule BAX is required for death of sympathetic and motor neurons in the setting of trophic factor deprivation. Furthermore, adult Bax-/- mice have more motor neurons than do their wild-type counterparts. These findings raise the possibility that BAX regulates naturally occurring cell death during development in many neuronal populations. To test this idea, we assessed apoptosis using TUNEL labeling in several well-studied neural systems during embryonic and early postnatal development in Bax-/- mice. Remarkably, naturally occurring cell death is virtually eliminated between embryonic day 11.5 (E11.5) and postnatal day 1 (PN1) in most peripheral ganglia, in motor pools in the spinal cord, and in the trigeminal brainstem nuclear complex. Additionally, reduction, although not elimination, of cell death was noted throughout the developing cerebellum, in some layers of the retina, and in the hippocampus. Saving of cells was verified by axon counts of dorsal and ventral roots, as well as facial and optic nerves that revealed 24-35% increases in axon number. Interestingly, many of the supernumerary axons had very small cross-sectional areas, suggesting that the associated neurons are not normal. We conclude that BAX is a critical mediator of naturally occurring death of peripheral and CNS neurons during embryonic life. However, rescue from naturally occurring cell death does not imply that the neurons will develop normal functional capabilities.

Animals↗
PubMed1998

Bcl-xL is an antiapoptotic regulator for postnatal CNS neurons.

Bcl-xL is a death-inhibiting member of the Bcl-2/Ced9 family of proteins which either promote or inhibit apoptosis. Gene targeting has revealed that Bcl-xL is required for neuronal survival during brain development; however, Bcl-xL knock-out mice do not survive past embryonic day 13.5, precluding an analysis of Bcl-xL function at later stages of development. Bcl-xL expression is maintained at a high level postnatally in the CNS, suggesting that it may also regulate neuron survival in the postnatal period. To explore functions of Bcl-xL related to neuron survival in postnatal life, we generated transgenic mice overexpressing human Bcl-xL under the control of a pan-neuronal promoter. A line that showed strong overexpression in brainstem and a line that showed overexpression in hippocampus and cortex were chosen for analysis. We asked whether overexpression of Bcl-xL influences neuronal survival in the postnatal period by studying two injury paradigms that result in massive neuronal apoptosis. In the standard neonatal facial axotomy paradigm, Bcl-xL overexpression had substantial effects, with survival of 65% of the motor neurons 7 d after axotomy, as opposed to only 15% in nontransgenic littermates. To investigate whether Bcl-xL regulates survival of CNS neurons in the forebrain, we used a hypoxia-ischemia paradigm in neonatal mice. We show here that hypoxia-ischemia leads to substantial apoptosis in the hippocampus and cortex of wild-type neonatal mice. Furthermore, we show that overexpression of Bcl-xL is neuroprotective in this paradigm. We conclude that levels of Bcl-xL in postnatal neurons may be a critical determinant of their susceptibility to apoptosis.

Animals↗
PubMed1998

GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys.

Glial cell line-derived neurotrophic factor (GDNF) signals through a receptor complex composed of the Ret tyrosine kinase and a glycosylphosphatidylinositol- (GPI-) anchored cell surface coreceptor, either GDNF family receptor alpha1 (GFR alpha1) or GFR alpha2. To investigate the usage of these coreceptors for GDNF signaling in vivo, gene targeting was used to produce mice lacking the GFR alpha1 coreceptor. GFR alpha1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both GDNF- and Ret-deficient mice. Midbrain dopaminergic and motor neurons in GFR alpha1 null mice were normal. Minimal or no neuronal losses were observed in a number of peripheral ganglia examined, including the superior cervical and nodose, which are severely affected in both Ret- and GDNF-deficient mice. These results suggest that while stringent physiologic pairing exists between GFR alpha1 and GDNF in renal and enteric nervous system development, significant cross-talk between GDNF and other GFR alpha coreceptors must occur in other neuronal populations.

Animals↗
PubMed1997

Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons.

Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is required for the survival of all small and many intermediate-sized murine dorsal root ganglion (DRG) neurons during development, accounting for 80% of the total DRG population. Surprisingly, NGF/TrkA-dependent neurons include a large population that does not express TrkA in adult mice (Silos-Santiago et al., 1995). This finding suggests two hypotheses: Neurons lacking TrkA in the adult may express TrkA during development, or they may be maintained through a paracrine mechanism by TrkA-expressing neurons. To determine whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood, we examined the distribution of TrkA protein during development. We show here that TrkA expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at postnatal day (P) 21. Because the period of TrkA down-regulation corresponds with a critical period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell [1993] Trends Neurosci. 16:353-359), we examined whether NGF modulates the down-regulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the extent of TrkA down-regulation. Our results demonstrate a novel form of regulation of neurotrophin receptor expression that occurs late in development. All DRG neurons that require NGF for survival express TrkA during embryogenesis, and many continue to express TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The subsequent down-regulation of TrkA is likely to be importantly related to functional distinctions among nociceptive neurons in maturity.

Aging↗
PubMed1997

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Amino Acid Sequence↗