Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (~ T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p 0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (approximately T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p < or =0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Transient expression of GAP-43 in nonneuronal cells of the embryonic chicken limb.

Growth associated protein (GAP)-43 is a membrane-bound phosphoprotein expressed in neurons and is particularly abundant during periods of axonal outgrowth in development and regeneration of the nervous system. In previous work, we cloned a full-length chicken GAP-43 cDNA and described the expression of its corresponding mRNA during early development of the chicken nervous system. We report here that the GAP-43 mRNA is also expressed transiently in developing limbs of chicken embryos, which contain axons of spinal cord and dorsal root ganglion neurons, but do not contain neuronal cell bodies. GAP-43 mRNA was first detectable by RNA blot analysis in limbs from Embryonic Day 5 (E5) embryos, reached maximal levels between E6 and E8, and diminished by E10. In situ hybridization analysis showed that the GAP-43 mRNA was localized in distal regions of developing limbs and was particularly abundant in the mesenchyme surrounding the digital cartilage. In some regions of the limb, GAP-43 immunoreactivity colocalized in cells that were also immunoreactive for meromyosin, a muscle-specific marker. These data suggest that both GAP-43 mRNA and the protein are expressed in nonneuronal cells of the developing limb, some of which may be part of the muscle cell lineage.     

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.     

IT Delivery of ChABC Modulates NG2 and Promotes GAP-43 Axonal Regrowth After Spinal Cord Injury

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8–2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.     

The 21-Aminosteroid U-74389F Attenuates Hyperexpression of GAP-43 and NADPH-Diaphorase in the Spinal Cord of Wobbler Mouse,a Model for Amyotrophic Lateral Sclerosis

The wobbler mouse suffers an autosomal recessive mutation producing severe neurodegeneration and astrogliosis in spinal cord. It has been considered a model for amyotrophic lateral sclerosis. We have studied in these animals the expression of two proteins, the growth-associated protein (GAP-43) and the NADPH-diaphorase, the nitric oxide synthesizing enzyme, employing immunocytochemistry and histochemistry. We found higher expression of GAP-43 immunoreactivity in dorsal horn, Lamina X, corticospinal tract and ventral horn motoneurons in wobbler mice compared to controls. Weak NADPH-diaphorase activity was present in control motoneurons, in contrast to intense labeling of the wobbler group. No differences in diaphorase activity was measured in the rest of the spinal cord between control and mutant mice. A group of animals received subcutaneously for 4 days a 50 mg pellet of U-74389F, a glucocorticoid-derived 21-aminosteroid with antioxidant properties but without glucocorticoid activity. U-74389F slightly attenuated GAP-43 immunostaining in dorsal regions of the spinal cord from wobblers but not in controls. However, in motoneurons of wobbler mice number of GAP-43 immunopositive neurons, cell processes and reaction intensity were reduced by U-74389F. The aminosteroid reduced by 50% motoneuron NADPH-diaphorase activity. Hyperexpression of GAP-43 immunoreactivity in wobbler mice may represent an exaggerated neuronal response to advancing degeneration or muscle denervation. It may also be linked to increased nitric oxide levels. U-74389F may stop neurodegeneration and/or increase muscle trophism and stop oxidative stress, consequently GAP-43 hyperexpression was attenuated. Wobbler mice may be important models to evaluate the use of antioxidant steroid therapy with a view to its use in human motoneuron disease.     

The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.     

Increased expression of GAP-43 in small sensory neurons after stimulation by NGF indicative of neuroregeneration in capsaicin-treated rats.

Intraplantar injections of human recombinant nerve growth factor (rhNGF-beta) into the hind paw of capsaicin-treated adult rats are known to lead to a recovery of depleted peptide transmitter substances, to the immunohistochemical reappearance of peptidergic innervation in the skin and in the dorsal horn of the spinal cord, as well as to a recovery of the function of capsaicin-lesioned neurons. In the present study a marker peptide for neuronal regeneration and outgrowth, growth associated protein 43 (GAP-43), was investigated in lumbar dorsal root ganglia (DRGs) and in the hindpaw skin, in order to differentiate which population of the sensory neurons responds with a neuroregenerative behaviour. In situ hybridization histochemistry (ISH) revealed that at day 8 after the capsaicin treatment GAP-43 expression was significantly increased in small DRG cells as compared to control animals, and treatment with NGF in capsaicinized rats lead to an even more pronounced increase of GAP-43 expression in the small-sized cell population. Intraepidermal labelling of GAP-43 peptide was observed in the skin of control animals, but was markedly reduced in the animals that were treated with capsaicin alone. However, intraepidermal GAP-43 immunoreactive (GAP-43-IR) fibres nearly fully recovered in the capsaicin + NGF-treated group. These results indicate that the population of small DRG cells shows spontaneous regenerative activity after a capsaicin lesion which does not lead to a successful recovery of nerve terminals in the skin. Only after an additional NGF treatment small DRG cells show an even stronger regenerative response which now also involves structural reorganization of neuron membranes and axogenesis in the periphery.     

Cloning, localization, and functional expression of sodium channel beta1A subunits

Auxiliary beta1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of alpha and beta1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of beta1, termed beta1A, that results from an apparent intron retention event. beta1 and beta1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. beta1A mRNA expression is developmentally regulated in rat brain such that it is complementary to beta1. beta1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of beta1 expression. In contrast, beta1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed beta1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of beta1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of alphaIIA and beta1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing alphaIIA alone. This increase in current density reflected two effects of beta1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [(3)H]Saxitoxin binding analysis revealed a 4-fold increase in B(max) with no change in K(D) in cells coexpressing alphaIIA and beta1A compared with cells expressing alphaIIA alone. beta1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of beta1A subunits on sodium channel function may be physiologically important events in the development of excitable cells.     

甘麦大枣汤对抑郁模型大鼠海马神经元突触结构及结构蛋白表达的影响*

目的: 观察甘麦大枣汤对抑郁模型大鼠行为学及单胺递质的影响,并从突触结构及结构蛋白MAP-2与GAP-43表达改变探讨其潜在作用机制。方法: 60只SD大鼠随机分为5组:正常对照组、模型组、氟西汀组(10.8 mg/kg)、甘麦大枣汤高、低剂量组(9.72、4.86 g/kg),每组12只。除对照组外,其余各组大鼠均采用慢性不可预见性温和应激(CUMS)建立抑郁模型,并于造模同时给药组灌胃给药,连续21 d。采用糖水消耗实验和旷场测试评价大鼠抑郁样行为,ELISA法检测海马单胺递质5-HT、NE含量,Golgi染色观察神经元突触损伤情况,免疫组化法和Western blot法检测海马突触结构蛋白MAP-2和GAP-43的表达。结果: 与对照组比较,抑郁模型大鼠糖水偏好度及自主活动评分均显著下降(P＜0.01),海马5-HT、NE含量显著下降(P＜0.01),树突棘缺失明显,同时MAP-2和GAP-43表达均显著下调(P＜0.01);甘麦大枣汤干预后,模型大鼠抑郁样行为明显缓解(P＜0.01),5-HT、NE含量显著升高(P＜0.05),树突棘密度、长度及分枝增加,MAP-2和GAP-43表达显著回升(P＜0.01)。结论: 甘麦大枣汤能改善抑郁模型大鼠的抑郁样行为,升高海马单胺递质含量,其作用可能与上调突触结构蛋白、缓解神经元突触损伤有关。     

Cellular basis of steroid neuroprotection in the wobbler mouse, a genetic model of motoneuron disease

1. The Wobbler mouse suffers an autosomal recessive mutation producing severe motoneuron degeneration and astrogliosis in the spinal cord. It has been considered a suitable model of human motoneuron disease, including the sporadic form of amyotrophic lateral sclerosis (ALS).2. Evidences exist demonstrating increased oxidative stress in the spinal cord of Wobbler mice, whereas antioxidant therapy delayed neurodegeneration and improved muscle trophism. 21-Aminosteroids are glucocorticoid-derived hydrophobic compounds with antioxidant potency 3 times higher than vitamin E and 100 times higher than methylprednisolone. They do not bind to intracellular receptors, and prevent lipid peroxidation by insertion into membrane lipid bilayers.3. In common with the spinal cord of ALS patients, Wobbler mice present astrocytosis with hyperexpression of glial fibrillary acidic protein (GFAP), and increased expression of nitric oxide synthase (NOS) and growth-associated protein (GAP-43) in motoneurons. Here, we review our studies on the effects of a 21-aminosteroid on GFAP, NOS, and GAP-43.4. First, we showed that 21-aminosteroid treatment further increased GFAP-expressing astrocytes in gray matter of the Wobbler spinal cord. This effect may provide neuroprotection if one considers a trophic and beneficial function of astrocytes during the course of degeneration. Other neuroprotectans used in Wobbler mice (T-588) also increased preexisting astrocytosis.5. Second, histochemical determination of NADPH-diaphorase, a parameter indicative of neuronal NOS activity, showed that the 21-aminosteroid down-regulated the high activity of this enzyme in ventral horn motoneurons. Therefore, suppression of nitric oxide by decreasing NADPH-diaphorase (NOS) activity may provide neuroprotection considering that excess NO is highly toxic to motoneurons.6. Finally, 21-aminosteroid treatment significantly attenuated the aberrant expression of both GAP-43 protein and mRNA in Wobbler motoneurons. Hyperexpression of GAP-43 possibly indicated abnormal synaptogenesis, denervation, and muscle atrophy, parameters which may return to normal following antioxidant steroid treatment.7. Besides 21-aminosteroids, other steroids also behave as neuroprotectans. In this regard, degenerative diseases may constitute potential targets of these hormones, based on the fact that the spinal cord expresses in a regional and cell-specific fashion, receptors for androgens, progesterone, adrenal steroids, and estrogens.     

Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Both types of cells express classical intracellular PROG receptors (PR), while neurons additionally express the PROG membrane-binding site called 25-Dx. In motoneurons from rats with spinal cord injury (SCI), PROG restores to normal the deficient levels of choline acetyl-transferase and of alpha3 subunit Na,K-ATPase mRNA, while levels of the growth associated protein GAP-43 mRNA are further stimulated. Recent studies suggest that neurotrophins are possible mediators of hormone action, and in agreement with this assumption, PROG treatment of rats with SCI increases the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels in ventral horn motoneurons. In situ hybridization (ISH) has shown that SCI reduces BDNF mRNA levels by 50% in spinal motoneurons, while PROG administration to injured rats (4mg/kg/day during 3 days, s.c.) elicits a three-fold increase in grain density. In addition to enhancement of mRNA levels, PROG increases BDNF immunoreactivity in perikaryon and cell processes of motoneurons of the lesioned spinal cord, and also prevents the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. Our findings strongly indicate that motoneurons of the spinal cord are targets of PROG, as confirmed by the expression of PR and the regulation of molecular parameters. PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection. Thus, PROG treatment constitutes a new approach to sustain neuronal function after injury.     

Phosphorylation-site mutagenesis of the growth-associated protein GAP- 43 modulates its effects on cell spreading and morphology

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane- association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP- 43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin- containing cortical cytoskeleton in a regulatable manner.     

Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.