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L I Benowitz

Publications and source records attributed to L I Benowitz.

At least 37 records · Page 2Linked to original sources
PubMed1995

Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture.

Unlike mammals, lower vertebrates can regenerate an injured optic nerve and other pathways of the CNS throughout life. We report here that in dissociated cell culture, goldfish retinal ganglion cells regenerate their axons in response to two factors derived from the sheath cells of the optic nerve. Axogenesis factor 1 (AF-1) is a small peptide (700-900 Da) that is inactivated by treatment with proteinase K but heat stable. A second factor, AF-2, is a polypeptide of ca 12 kDa. In the absence of these factors, dissociated retinal cells remained viable in serum-free, defined media for at least a week but showed little outgrowth, as visualized using the vital dye 5,6-carboxyfluorescein diacetate (5,6-CFDA). The addition of AF-1 induced up to 25% of cells in culture to extend processes > 75 microns in length by 6 d; AF-2 had a lesser but highly significant effect. To verify that neurite outgrowth was from retinal ganglion cells per se, we applied the lipophilic dye 4-Di-10-ASP to the optic tectum and allowed it to diffuse up the optic nerve for several days before culturing the retina. A far greater percentage of cells containing the dye showed axonal outgrowth than was observed from the overall cell population, indicating that ganglion cells are selective targets of the factors. The effects of AF-1 or AF-2 were not secondary to enhanced viability, since neither overall cell survival nor the number of retinal ganglion cells remaining in culture after 6 d was affected by the presence of the factors. The activity of AF-1 and AF-2 was not mimicked by several defined factors tested over a broad concentration range, for example, NGF, BDNF, NT-3, CNTF, taurine, retinoic acid, acidic or basic fibroblast growth factors. The concentration of AF-1 is considerably higher in CM than in optic nerve homogenates, suggesting that it is actively secreted; AF-2 has a similar concentration intra- and extracellularly. Insofar as AF-1 and AF-2 derive from cells of the optic nerve and act upon retinal ganglion cells, they are likely to be important in inducing optic nerve regeneration in vivo.

Animals↗
PubMed1994

The amyloid precursor protein is developmentally regulated and correlated with synaptogenesis.

Metabolism of the amyloid precursor protein (APP) may contribute to the molecular changes observed in Alzheimer's disease, but the function of the protein in the non-pathologic nervous system remains unknown. In vitro studies have suggested that APP can participate in cellular adhesion and may thus contribute to neuronal differentiation in cultured cells. Here we show, in the primary visual pathway of the hamster, that APPs are developmentally regulated proteins rapidly transported to the growing tips of nerve fibers. Transmembrane forms of higher molecular weight (120 and 140 kDa) are preferentially associated with the rapid elongation of axons. Interestingly, another full-length form of 110 kDa and a soluble form of 100 kDa which lacks the C-terminal domain increase at the time of end-arbor formation and synaptogenesis and then decline when mature connections are established, suggesting that target recognition and synaptic contact may result in a signal for APP cleavage in the CNS in vivo.

Age Factors↗
PubMed1994

Injury induced expression of growth-associated protein-43 in adult mouse retinal ganglion cells in vitro.

In optic fibers, as in most axons of the central nervous system, the axonal growth-associated protein, GAP-43, is abundant during development but absent in adults. Since optic fibers can be induced to regenerate in culture, we examined whether this was associated with an increased expression of GAP-43 in adult mouse optic fibers that were regenerating from organotypic retinal explants on to laminin substrates. We found that simply placing adult mouse retina in culture under serum-free conditions was sufficient to induce GAP-43, which was detectable after about four to five days in vitro, coincident with the initiation of neurite outgrowth. In explants taken from animals in which the optic nerve was crushed in the orbit eight days prior to culturing, GAP-43 was observed within one day, as was neurite outgrowth. This priming effect was also seen in vivo as an increased level of GAP-43 reactivity in retinal ganglion cells and optic fibers in histological sections taken eight days after nerve crush. Reactivity in the adult fibers in culture was comparable to that observed in optic neurites growing from embryonic retinal explants and could be maintained for at least four weeks in culture. In the adult neurites, especially with longer times in culture, GAP-43 tended to be concentrated into varicosities that were often found in terminal-like arbors that formed in culture. Placing adult retina in culture under serum-free conditions in sufficient to induce re-expression of GAP-43 for an indefinite period of time. This suggests that GAP-43 expression and the propensity for growth in vivo may be repressed by a factor that is absent in vitro.

Aging↗
PubMed1994

Innervation of the rat anterior and neurointermediate pituitary visualized by immunocytochemistry for the growth-associated protein GAP-43.

Immunocytochemical localization of the neuronal growth associated protein GAP-43 revealed a dense axonal plexus throughout the neurointermediate lobe of the rat pituitary. These axons were fine, presumably monoaminergic fibers, whereas magnocellular neurosecretory axons did not appear to contain detectable GAP-43. These experiments also revealed the presence of an extensive nerve plexus within the anterior lobe. Fine beaded fibers were present throughout the parenchyma of the anterior lobe, and punctate staining suggestive of nerve terminals was seen surrounding numerous endocrine cells. Nerve fibers did not appear to cross directly between the intermediate and anterior lobes, but rather entered the anterior lobe directly from its margins or in association with blood vessels. Preabsorption of antisera with GAP-43 purified from neonatal rat brain completely eliminated immunoreactivity. These findings confirm the existence of a direct innervation of the anterior pituitary of the rat; moreover, the presence of GAP-43 in these fibers suggests that they may be capable of growth and terminal reorganization in the adult animal.

Animals↗
PubMed1994

GAP-43 expression in primary sensory neurons following central axotomy.

Primary sensory neurons are capable of successful regenerative growth in response to peripheral nerve but not dorsal root injury. The present study is concerned with the differential expression of the mRNA for GAP-43, a growth-associated protein, in these sensory neurons, in response to injury of their central or peripheral axonal branches. Peripheral axotomy resulted in an elevation in message detectable within 24 hr, using Northern blot and in situ hybridization, which was maintained for 30 d, whereas dorsal root section produced no change except a transient and small increase if the axotomy was immediately adjacent to the dorsal root ganglia (DRG). Dorsal root section had no effect on GAP-43 mRNA levels in the dorsal horn or in neighboring intact DRG. It also failed to alter the laminar boundaries of the GAP-43 central terminal labeling produced by peripheral nerve section, even though vacant synaptic sites were produced in unstained laminae by this procedure. This indicates that the location of GAP-43 immunolabeling in the central terminals of primed sensory cells may not depend only on the location of vacant synaptic sites. We conclude that distinct control mechanisms regulate the response of DRG neurons to peripheral nerve and dorsal root injury, and these may be related both to the glial environment and the particular target influences exerted on the central and peripheral branches of the primary sensory neuron. Central denervation alone is insufficient to upregulate GAP-43 levels, and this may explain the relative absence of collateral sprouting after the production of central vacant synaptic sites. The failure of dorsal root section to increase GAP-43 expression may contribute to the poor regenerative response initiated by such lesions.

Animals↗
PubMed1993

Anatomic distribution of the growth-associated protein GAP-43 in the developing human brainstem.

GAP-43 is a membrane phosphoprotein whose expression is high in neurons undergoing development or remodeling of axonal connections. This study used a monospecific antibody to GAP-43 to investigate the sequences of fiber tract elongation and synaptic development in the human brainstem. Immunocytochemistry was performed in 14 fetal and infant brainstems; two child and adult cases were also examined for comparison. At midgestation, GAP-43 immunostaining was moderately intense across nuclei and fiber tracts, except for the corticospinal tract, where levels were higher, and cranial nerve nucleus VII, superior olive, inferior colliculus, inferior olivary hilum, inferior cerebellar peduncle, medial lemniscus, and medial longitudinal fasciculus, where staining was nearly absent. By the end of the neonatal period, the relative distribution of GAP-43 immunostaining appeared well-established and similar, although not identical, to that in the child and adult brainstem. Immunostaining was absent or negligible in almost all the cranial nerve somato- and branchiomotor nuclei, auditory-relay nuclei, and vestibular nuclei, while remaining intense in visceral-related nuclei, reticular formation, cochlear nucleus, and periaqueductal gray. Staining was also virtually absent in all fiber tracts at birth, except for the corticospinal tract and central tegmental tract. Persistence of GAP-43 staining in the corticospinal tract past the fetal period suggests that this tract remains in a plastic state beyond initial axonal elongation. Intense immunostaining in visceral-related nuclei into adulthood suggests that these regions may continue to undergo synaptic reorganization. This study provides baseline information relevant to understanding developmental brainstem disorders in early human life.

Brain Stem↗
PubMed1993

Activation of protein kinase C by arachidonic acid selectively enhances the phosphorylation of GAP-43 in nerve terminal membranes.

Arachidonic acid (AA), a cis-unsaturated fatty acid that activates certain subspecies of protein kinase C (PKC), has been proposed to act as a retrograde messenger in modifying the efficacy of synapses during long-term potentiation (LTP). One prominent PKC substrate of the nerve terminal membrane, GAP-43 (F1, B-50, neuromodulin), shows an increase in phosphorylation that correlates with the persistence of LTP. The present study investigated whether AA might exert its effects on presynaptic endings by modulating the phosphorylation of GAP-43 and other membrane-bound proteins. Using synaptosomal membranes from the rat cerebrocortex, in which in vivo relationships between protein kinases and their native substrates are likely to be preserved, we found that in the absence of Ca2+, AA exerted a modest effect on the phosphorylation of GAP-43 and several other proteins; however, when AA was applied in conjunction with Ca2+, GAP-43 showed a particularly striking response: at Ca2+ levels likely to exist at the nerve terminal membrane during synaptic activity (10(-7) to 10(-5) M), AA (50 microM) increased the sensitivity of GAP-43 phosphorylation to Ca2+ by an order of magnitude, and increased its maximal level of phosphorylation by 50%. At resting Ca2+ levels, AA potentiated the stimulation in GAP-43 phosphorylation produced by 4 beta-phorbol 12,13-dibutyrate, a diacylglycerol (DAG) analog. The stimulatory effect of AA and its synergistic interaction with Ca2+ were found to be mediated by PKC, since they were blocked by a specific peptide inhibitor of PKC, [Ala25]PKC(19-31), but were unaffected by an inhibitor of protein phosphatase activity or by scavengers of free radicals. Since GAP-43 has been implicated in the development and plasticity of synaptic relationships, the synergistic effects of AA and the intracellular signals Ca2+ and DAG on the phosphorylation of GAP-43 may serve as an AND gate to modify presynaptic function and/or structure in response to coincident pre- and postsynaptic activity.

Animals↗
PubMed1992

Changes in rapidly transported proteins associated with development of abnormal projections in the diencephalon.

The development of the hamster visual system is accompanied by striking changes in the pattern of proteins that are synthesized in retinal ganglion cells and rapidly transported to their nerve terminals. To determine whether any of these protein changes are regulated by interactions between the developing nerve endings and the cells with which they form synapses, we induced retinofugal axons to form abnormal projections in the lateral posterior (LP) nucleus of the thalamus and dense patches of hyperinnervation in the lateral geniculate nucleus (LGN) by removing their principal target, the superior colliculus (SC), the day after birth. Under these experimental conditions, two rapidly transported proteins, including the neural cell adhesion molecule, NCAM, showed significant changes in their time course of expression. NCAM, identified here using a monospecific antibody, is normally synthesized and transported at high levels at early stages of development and then declines during the second and third postnatal weeks. However, this decline was delayed when optic fibers were re-routed. A second rapidly transported protein, M(r) = 67 kDa, pI = 4.7, normally shows a rise in its synthesis and transport during terminal arbor formation and a subsequent decline, but it also remained elevated for a prolonged period when the SC was absent. These findings cannot be accounted for by a simple delay in the retinal ganglion cells' program of axonal growth, since other rapidly transported proteins, including the growth-associated protein GAP-43, showed a normal developmental time-course when the SC was removed. Target interactions therefore appear to influence the retinal ganglion cells' expression of different proteins in a specific fashion.

Aging↗
PubMed1992

Vibrissectomy induced changes in GAP-43 immunoreactivity in the adult rat barrel cortex.

Within the rat primary somatosensory cortex, neurons responding principally to movement of each individual mystacial vibrissa are grouped together in structures termed barrels. Previous studies have examined changes in the area of cortex showing increased 2-deoxyglucose uptake in response to vibrissal stimulation. These studies have shown that chronic removal of all but the central (C3) vibrissa in adult rats induces an enlarged representation of the remaining C3 barrel in the contralateral cortex. This increase is prevented by cortical norepinephrine depletion. The major question raised by such studies is whether such plasticity is due to structural rearrangement or unmasking of otherwise silent synapses. In this study, antibodies to GAP-43, a presynaptic protein whose synthesis is related to neuronal development and regeneration, were used to investigate this issue. In adult rat brain, tangential sections through layer IV of the barrel receptor field normally show moderate levels of GAP-43 immunoreactivity (GAP-IR) in the inter-barrel septa and low levels within the barrels themselves. The present study examined changes in the pattern of GAP-IR from 1 to 8 weeks after vibrissectomy with sparing of C3 as an index of possible physical reorganization of cortical circuits. Quantitative analysis of the cortices of animals with unilateral vibrissectomy with sparing of C3 showed that the area of low GAP-IR within the barrels surrounding C3 was decreased at 1 week (8.4% shrinkage; P less than 0.01) and 8 weeks (12.0% shrinkage; P less than 0.015), relative to the cortex ipsilateral to the surgery. Both bilateral vibrissectomy with sparing of C3 and ibotenic acid lesions of the ventrobasal thalamus produced similar results. Some evidence was also seen that the area of low GAP-IR in the C3 barrel shrank to a similar degree after such manipulations. Cortical norepinephrine depletion had no apparent effect on vibrissectomy-induced GAP-IR changes. These results suggest that removal of vibrissal input to the adult rat barrel cortex produces transynaptic induction of axonal sprouting within the barrel cortex.

Afferent Pathways↗
PubMed1992

Changes in chromatin proteins during optic nerve regeneration in the goldfish.

Regeneration of the goldfish optic nerve involves massive changes in the structure and pattern of macro-molecular synthesis in the retinal ganglion cells. To explore the mechanisms that underlie these events, we investigated the changes in chromatin proteins during the course of regeneration. Three major retinal chromatin proteins, two with apparent molecular weights of 58 kDa (C1 and C2) and one at 51 kDa (C3), all having isoelectric points around 5.5, showed a fourfold increase in their synthesis and/or accumulation by 14 days of regeneration. Synthesis of C1 and C3 decreased by day 32, the time at which the axons have grown back to the optic tectum and have formed many of their synapses; synthesis of C2 remained high through day 32. All three proteins bound to DNA-cellulose and required high salt concentrations (0.2-0.5 M KCl) to be eluted. C1 and C2 had similar proteolytic digestion patterns and reacted with monoclonal antibodies that recognize the goldfish intermediate filament proteins of the ON complex. The proteins identified here could be involved in structural alterations in the chromatin, or might serve as transcription factors to regulate gene expression during nerve regeneration.

Animals↗
PubMed1992

Denervation of the motor endplate results in the rapid expression by terminal Schwann cells of the growth-associated protein GAP-43.

Developing and regenerating neurons express high levels of the growth-associated phosphoprotein GAP-43. This membrane protein is not confined to neurons, however, as a number of studies have demonstrated GAP-43 immunoreactivity in central and peripheral glia in vitro and in vivo. We have found that the Schwann cells overlying the terminal motor axon at adult rat skeletal muscle endplates, and the motor axons themselves, are normally not GAP-43 immunoreactive. Within 24 hr of denervation, however, the terminal Schwann cells are positive for a GAP-43 mRNA in situ hybridization signal and are GAP-43 immunoreactive. The immunoreactive GAP-43 cells possess elaborate processes that branch from the endplate region into the perisynaptic zone and stain with defined Schwann cell markers: the calcium binding protein S100 and the low-affinity NGF receptor (NGFr), but not with a fibroblast marker, Thy-1. Reinnervating motor axons are GAP-43 positive, with an appearance quite different from the GAP-43-positive Schwann cells. The reappearance of nerve endings at the motor endplate is followed by the disappearance of GAP-43 labeling in the Schwann cells and of a retraction of their processes. GAP-43 expression in Schwann cells is therefore state dependent, apparently regulated by neural contact. This protein, which is associated in neurons with neurite formation, may participate in the elaboration of processes by Schwann cells when their contact with axons is disrupted.

Animals↗
PubMed1991

Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention.

GAP-43 is a presynaptic membrane phosphoprotein that has been implicated in both the development and the modulation of neural connections. The availability of cDNA clones for GAP-43 makes it possible to examine with greater precision its role in neuronal outgrowth and physiology. We used Northern blots and in situ hybridization with GAP-43 antisense RNA probes to show that GAP-43 is expressed selectively in associative regions of the adult brain. Immunocytochemical analyses showed alterations in the pattern of GAP-43 expression in the hippocampus during reactive synaptogenesis following lesions of the perforant pathway. Genetic intervention methodology was used to analyze the molecular nature of GAP-43 involvement in synaptic plasticity. GAP-43-transfected PC12 cells displayed an enhanced response to nerve growth factor, suggesting that GAP-43 may be directly involved in neurite extension and in the modulation of the neuronal response to extrinsic trophic factors. Studies of PC12 cell transfectants, in which the synthesis of GAP-43 was blocked by expression of GAP-43 antisense RNA, showed that evoked dopamine release was significantly attenuated in these cells. The use of gene transfer into neurons with the HSV-1 vector is presented as a method of analyzing the interaction of GAP-43 with signal transduction systems during neurotransmitter release.

Animals↗
PubMed1991

Mapping the development of the rat brain by GAP-43 immunocytochemistry.

Growth-associated protein-43 (GAP-43) is a phosphoprotein of the nerve terminal membrane which has been linked to the development and restructuring of axonal connections. Using a monospecific antibody prepared in sheep against purified GAP-43, we examined the temporal and spatial changes in the distribution of this protein from embryonic stage day 13 (E13) to adulthood. At stages in which neurons are still dividing and migrating, levels of GAP-43 are extremely low, as is seen in the cortical plate throughout the embryonic period. With the onset of process outgrowth, intense GAP-43 immunoreactivity appears along the length of axons: by E13, such staining is already strong in the brainstem, where it continues up through the first postnatal week and then disappears. In the neocortex, intense fiber staining first appears several days later but ends at the same time as in the brainstem. At the end of the period of intense axonal staining there is a brief interval in which high levels of GAP-43 immunostaining are seen in the neuropil. In regions of the brain in which specific developmental events have been characterized anatomically and physiologically, the period of dense neuropil staining coincides with the formation of axonal end-arbors, the beginning of synaptogenesis, and the time at which synaptic organization can be modified by the impingent pattern of activity (i.e. the critical period). Over the next few days, staining in neuropil declines sharply in most regions except for certain structures in the rostral neuraxis which may be sites of ongoing synaptic remodeling.

Animals↗
PubMed1991

GAP-43 expression in the developing rat lumbar spinal cord.

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.

Animals↗
PubMed1991

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.

Animals↗
PubMed1991

Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis.

The neuronal growth-associated protein GAP-43 is expressed during axonal outgrowth and regeneration (for review, see Benowitz and Routtenberg, 1987). In the present study, we demonstrate that GAP-43 is constitutively expressed by NB2a/d1 neuroblastoma cells. The initial, most rapid outgrowth period of neuritogenesis [0-4 hr after dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) treatment] is accompanied by intense GAP-43 immunoreactivity along the entire length of most neurites. However, this immunoreactivity declined nearly to background levels within hours during continued neurite outgrowth and persisted only at varicosities and growth cones. GAP-43 was detectable by metabolic labeling and immunoblot analysis in undifferentiated cells, and synthetic rates and steady-state levels of GAP-43 underwent only a modest (approximately twofold) increase during dbcAMP-induced differentiation. Unlike levels observed in neurites, perikarya of undifferentiated and differentiated cells contained similar, intense levels of GAP-43 immunoreactivity. Neurite elaboration and GAP-43 immunoreactivity were unaffected by treatment with cycloheximide, suggesting that translocation of perikaryal GAP-43 pools, rather than de novo synthesis, contributes to the transient burst of GAP-43 observed in developing neurites. Phosphatidylcholine-mediated delivery of anti-GAP-43 antibodies (alpha GAP) into cells immediately before dbcAMP treatment arrested neuritogenesis but did not induce the retraction of existing neurites. These results indicate that, while GAP-43 expression is insufficient to induce neuritogenesis in NB2a/d1 cells, GAP-43 is nevertheless essential for the initial, dynamic phase of neurite outgrowth.

Animals↗