Increase In GAP-43 Research Articles

Single vs. Combined Therapeutic Approaches in Rats With Chronic Spinal Cord Injury.

The regenerative capability of the central nervous system is limited after traumatic spinal cord injury (SCI) due to intrinsic and extrinsic factors that inhibit spinal cord regeneration, resulting in deficient functional recovery. It has been shown that strategies, such as pre-degenerated peripheral nerve (PPN) grafts or the use of bone marrow stromal cells (BMSCs) or exogenous molecules, such as chondroitinase ABC (ChABC) promote axonal growth and remyelination, resulting in an improvement in locomotor function. These treatments have been primarily assessed in acute injury models. The aim of the present study is to evaluate the ability of several single and combined treatments in order to modify the course of chronic complete SCI in rats. A complete cord transection was performed at the T9 level. One month later, animals were divided into five groups: original injury only (control group), and original injury plus spinal cord re-transection to create a gap to accommodate BMSCs, PPN, PPN + BMSCs, and PPN + BMSCs + ChABC. In comparison with control and single-treatment groups (PPN and BMSCs), combined treatment groups (PPN + BMSCs and PPN + BMSCs + ChABC) showed significative axonal regrowth, as revealed by an increase in GAP-43 and MAP-1B expression in axonal fibers, which correlated with an improvement in locomotor function. In conclusion, the combined therapies tested here improve locomotor function by enhancing axonal regeneration in rats with chronic SCI. Further studies are warranted to refine this promising line of research for clinical purposes.

Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush.

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.

Effects of developmental perfluorooctane sulfonate exposure on spatial learning and memory ability of rats and mechanism associated with synaptic plasticity

The present study aims to explore the effects of perfluorooctane sulfonate (PFOS) on cognitive function in developing rats and the underlying mechanism associated with synaptic plasticity. Pregnant Wistar rats were fed with 0, 5, and 15 mg/L of PFOS via drinking water during gestation and lactation. Offspring were exposed to PFOS on prenatal and/or postnatal days by cross-fostering. Spatial learning and memory abilities were tested from postnatal day (PND) 35. We also analyzed the expression pattern of the synaptic plasticity-related genes and proteins in the hippocampus on PND7 and PND35. Results revealed that PFOS exposure reduced the spatial learning and memory abilities of the offspring, particularly of those with prenatal exposure. Meanwhile, protein levels of growth-associated protein-43, neural cell adhesion molecule 1, nerve growth factor, and brain-derived neurotrophic factor decreased on PND35, which are involved in the formation of synaptic plasticity. In contrast, significant increase in gap-43, ncam1, and bdnf genes on the mRNA level was observed on PND7, possibly due to the post-transcriptional mechanism. Results of both behavioral effects and molecular endpoints suggested the high risk of prenatal PFOS exposure. The decline of spatial learning and memory abilities induced by developmental PFOS exposure was closely related to synaptic plasticity.

GAP‐43 immunoreactivity of subepithelial and detrusor muscle nerve fibres in patients with refractory idiopathic detrusor overactivity

To determine the relative density of nerve fibres immunoreactive to growth associated protein-43 (GAP-43, an indicator of neuronal sprouting) in the subepithelium and detrusor of patients with idiopathic detrusor overactivity (IDO). To investigate the effect, if any, of age and previous recurrent bacterial cystitis on neuronal sprouting in such patients. A series of 18 women with urodynamically proven IDO (median age 62 years, range 39-85), who were refractory to treatment, underwent cystoscopy and cold cup biopsy. Controls (n=26, median age 65, range 32-79) were females without urgency/urge incontinence, undergoing cystoscopy for other indications. Recurrent proven bacterial cystitis (rUTI) was documented. Frozen sections were stained with specific antibodies to GAP-43 and protein gene product 9.5 (PGP, a general neuronal marker). The area represented by immunoreactive (ir) subepithelial or muscle nerve fibres was measured. The density of GAP-43ir and PGPir nerves did not differ significantly between IDO patients and controls, in either subepithelium or detrusor. The GAP-43ir nerve density (as percent of PGPir) increased significantly with advancing age amongst patients with IDO in the detrusor muscle but not in the subepithelium; density in controls was unaltered. In IDO patients with rUTI, a significant increase in GAP-43 (as percent of PGPir) was observed in the subepithelium. Although we found no evidence of increased neuronal proliferation in patients with IDO generally, the increase in GAP-43 with age and with previous cystitis history suggests that neuronal sprouting is important in some subsets of patients with IDO.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro.

Matrix metalloproteinase-9 (MMP-9) is a basal-lamina-degrading protease that we have recently shown to be localized in regenerating sciatic nerve. We now demonstrate that MMP-9 colocalizes with growth-associated protein GAP-43 in regenerating nerves in vivo and is involved in vitro in axonal sprouting. By using a PC12 cell model for neuronal sprouting, we analyzed the effects of recombinant MMP-9, MMP-9-neutralizing antibody, and a broad-spectrum MMP inhibitor (Ro 31-9790) on sprout formation, elongation, and branching. Quantitative phase-contrast microscopy showed that MMP-9 elongated neuronal sprouts by 67% and increased their branching by 14% but did not change the number of sprouts relative to nerve growth factor (NGF) treatment. Double immunofluorescence for GAP-43, a marker for growth cones, and alpha-tubulin, a marker for axonal microtubules, showed that MMP-9-treated cells had increased distribution of alpha-tubulin but no effect on GAP-43. Western blot analyses of cell lysates demonstrated that the NGF-induced increase in GAP-43 was unchanged with MMP-9 treatment or inhibition, confirming that MMP-9 had no effect on new sprout formation. However, Ro 31-9790 reduced GAP-43 levels to those seen in untreated cells, suggesting that an MMP other than MMP-9 is important for sprout formation. Finally, phosphorylated neurofilament M (NFM-p), a marker for regenerative elongation, was induced with MMP-9 treatment and was inhibited by the anti-MMP-9 antibody treatment, confirming the role of MMP-9 in axonal elongation. NFM-p colocalized with MMP-9 in regenerating sciatic nerve fibers. These findings suggest that MMP-9 regulates neurite extension in regenerating peripheral nerve fibers and, therefore, might be of therapeutic value in promoting regeneration in vivo.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Parallel compensatory and pathological events associated with tau pathology in middle aged individuals with Down syndrome.

Aged individuals with Down syndrome (DS) develop senile plaques and neurofibrillary tangles consistent with Alzheimer disease (AD). Prior to or in parallel with AD pathology, compensatory growth responses may occur. Immunohistochemistry and confocal microscopy studies in the hippocampus from 15 individuals ranging in age from 5 months to 67 years compared markers of normal and abnormal tau accumulation (phosphorylated tau [AT8, MC-1], tau-1, N-terminal tau) with the extent and location of neuronal growth marker immunoreactivity (BDNF, GAP-43, MAP-2). In middle age (30-40 years), prior to entorhinal neuron loss, the earliest tau accumulation occurred in the outer molecular layer (OML), which was consistent with both pathological and compensatory fetal tau expression. These events were followed at a later age, associated with entorhinal neuron loss, by an increase in GAP-43. Hilar neurons exhibiting a sprouting morphology were also noted. Age-dependent observations in the DS brain in the current study parallel hippocampal compensatory responses described in entorhinal cortex lesion studies in rodents. Thus, compensatory growth responses may occur in DS prior to extensive AD pathology and may be one mechanism underlying observations in PET studies of hypermetabolism in the entorhinal cortex of individuals with DS.

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium.

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.

Overexpression of HuD Accelerates Neurite Outgrowth and Increases GAP-43 mRNA Expression in Cortical Neurons and Retinoic Acid-Induced Embryonic Stem Cells in Vitro

The neuron-specific RNA-binding protein HuD binds to a U-rich regulatory element of the 3′ untranslated region (3′ UTR) of the GAP-43 mRNA and stabilizes the mRNA. We have previously shown that overexpression of HuD in PC12 cells increases GAP-43 protein expression and induces the spontaneous formation of multiple neurites (K. D. Anderson et al. 2000. J. Neurochem. 75: 1103–1114). In this study, we examined the effects of HuD overexpression on the initial stages of neurite outgrowth and on GAP-43 gene expression using two in vitro systems: E19 rat cortical neurons and retinoic acid (RA)-induced embryonic stem (ES) cells. Normal neurite outgrowth of cortical neurons in vitro occurs over a 3-day period with a concomitant increase in GAP-43 and HuD expression. Cortical cells were infected with a replication-deficient HSV-1 vector containing the HuD cDNA in the sense orientation (HSV-HuD). Overexpression of HuD accelerated the formation of neurites. Immunocytochemical analysis showed that excess HuD resulted in a threefold increase in the number of GAP-43-positive cells undergoing morphological differentiation after 24 h of treatment. Using in situ hybridization, we found that the increased HuD expression resulted in a twofold increase in the levels of GAP-43 mRNA. Similarly, overexpression of HuD in RA-induced embryonic stem cells was found to increase the number of GAP-43-positive cells undergoing process outgrowth. In conclusion, our results demonstrate that HuD functions in the initiation of neurite outgrowth in a manner due, at least in part, to its regulation of GAP-43 expression.

Cyclic AMP Prevents an Increase in GAP-43 but Promotes Neurite Growth in Cultured Adult Rat Dorsal Root Ganglion Neurons

High expression of the growth-associated protein GAP-43 in neurons is correlated with developmental and regenerative axon growth. It has been postulated that during development and after injury, GAP-43 expression is elevated due to the unavailability of a target-derived repressive signal, but that GAP-43 expression then declines upon target contact. Here we examine the cyclic AMP second messenger signaling pathway to determine if it might mediate retrograde transmission of a signal which represses GAP-43 expression and inhibits growth. Cultures of adult rat dorsal root ganglia were chronically exposed to membrane-permeable analogs of cyclic AMP and activators of adenyl cyclase. These treatments caused GAP-43 protein levels to decrease in a dose-dependent manner, although neuronal survival was not affected. GAP-43 mRNA was also decreases by cyclic AMP. GAP-43 protein levels were not repressed by neurotrophins, cytokines, or other agents. Surprisingly, cyclic AMP caused an increase in the rate of neurite outgrowth, even though the neurons were partially depleted of GAP-43. Growth stimulation was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern. These findings suggest that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP.

Semiquantitative analysis of low levels of mRNA expression from small amounts of brain tissue by nonradioactive reverse transcriptase-polymerase chain reaction

We have developed an easy and fast method to semiquantify low levels of mRNA from small amounts of brain tissues based on nonradioactive reverse transcription-polymerase chain reaction (RT-PCR). The regulation of mRNA for the growth associated protein GAP-43/B-50 and the homeodomain protein islet-1 was examined in the facial nucleus of the rat after a unilateral transection of the nerve. In both cases a similar sensitivity for radioactive and nonradioactive RT-PCR methods was found. The expression of the housekeeping gene, cyclophilin A was used to normalize total mRNA amounts and PCR conditions. After amplification the PCR products were separated electrophoretically on polyacrylamide gels. For nonradioactive semiquantification gels were stained with ethidium bromide and recorded using a CCD camera and transillumination. The recordings were evaluated with specialized software. Using nonradioactive RT-PCR, the increase in GAP-43/B-50 mRNA in response to axotomy was easily detectable in the small volume of tissue obtained from the facial nucleus. In contrast, the low expression of islet-1 mRNA made it necessary to develop a two-step amplification procedure in order to provide a reliable semiquantitative analysis. The procedure included preamplification of the cDNA and subsequent purification of the cDNA. Using this method, the down-regulation of islet-1 could be demonstrated with a similar sensitivity to that previously shown with radioactive RT-PCR.

Increase in GAP-43 and GFAP immunoreactivity in the rat hippocampus subsequent to perforant path kindling.

Kindling is an animal model of epilepsy which is accompanied by morphological and biochemical changes in the brain, including sprouting of fibers and increased transmitter release. Here we have examined the immunocytochemical expression of 1) GAP-43, a growth-associated protein, which is a neuron-specific PKC substrate, particularly expressed in development and regeneration and 2) glial fibrillary acidic protein (GFAP), part of the astrocytic cytoskeleton, after perforant path kindling. Subsequent to kindling, GAP-43 immunoreactivity was increased in CA1 stratum lacunosum-moleculare and the inner and outer molecular layer of the fascia dentata. Other hippocampal subregions showed a lower increase. GFAP immunoreactivity was increased in the entire hippocampus, but especially in stratum lacunosum-moleculare of the CA1 and the hilus of fascia dentata. The difference between the number of GFAP-positive profiles in the hippocampus of control rats and in fully kindled rats was found to be non-significant. We interpret these findings as being related to both plastic neuronal changes and possible neuronal degeneration.

Neuronal sprouting in mouse sensory ganglia infected with herpes simplex virus type 2 (HSV-2): induction of growth-associated protein (GAP-43) and ultrastructural evidence.

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.

The consequences of microneurography electrode-induced injury of peripheral nerves observed in the rat and man

The consequences of microneurography were assessed, using tungsten and coaxial electrodes in rat and man. Firstly, the sequelae of microneurography were examined for up to 1 month in a rat sciatic nerve model, using 3 techniques. Expression of the injury-associated protein GAP-43 was measured by immunofluorescence in sciatic nerve and dorsal root ganglia 3 and 28 days following sciatic nerve injury by microneurography electrodes. No increase in GAP-43 was observed in sciatic nerve 3 days following injury, but 28 days after injury the coaxial electrode was associated with an increase in GAP-43 expression. All electrodes were associated with an increase in GAP-43 in dorsal root ganglia 28 days after injury. The capacity of unmyelinated afferent fibres to induce neurogenic oedema was examined up to the 28th post-experimental day, as measured by Evan's Blue extravasation. The tungsten electrode induced a decrease in extravasation, which persisted for 28 days. In contrast, the decrease in plasma extravasation associated with lesions induced by the coaxial electrode returned to baseline within 7 days. The hind-limb withdrawal time from a noxious stimulus was also measured up to 28 days following injury. Both types of electrode induced a significant change in the immediate post-experimental period, but this returned to normal within 1 week. The direction of change differed between the tungsten and coaxial electrodes. In humans, symptomatology questionnaires were collected after microneurography experiments with the coaxial electrode. Thirty-two volunteers were studied. Two subjects reported mild paraesthesiae, which fully resolved within 24 h. This compares favourably with similar studies of the tungsten electrode.

The Recovery of Postural Reflexes and Locomotion Following Low Thoracic Hemisection in Adult Cats Involves Compensation by Undamaged Primary Afferent Pathways

Spinal hemisection in the adult cat results in motor impairments followed by substantial recovery of function (16, 20, 39, 53). The present study was undertaken to assess the contribution of undamaged ipsilateral segmental and contralateral descending systems to recovery of motor function. Quantitative behavioral methods were used to examine monopedal reflex and bipedal locomotor functions after thoracic hemisection. Different facets of motor behavior recover at different times. The recovery of monopedal postural reflexes precedes the recovery of more complex motor behavior. Since the reflexes tested are initiated by segmental afferent input and show recovery and normal motor patterns during locomotion, as defined by kinematic analysis show recovery, it is likely that dorsal root input compensates for the loss of descending input to one side of the spinal cord. Quantitative immunocytochemical methods for visualizing the central projections of dorsal root fibers (monoclonal antibody RAT-102; 49) and the descending serotoninergic pathway were used to examine the response of these pathways to hemisection. Hemisection results in a permanent decrease in the density of serotoninergic projections and a permanent increase in dorsal root projections in the spinal cord. The increased density of RAT-102 may represent an increase in the projection of dorsal root fibers and provide the increased input necessary to mediate enhanced reflex control. A transient increase in GAP-43 in the dorsal horn ipsilateral to the hemisection suggests that the increased density of RAT-102 immunoreactivity is associated with growth. Taken together, our results suggest that sprouting of primary afferents within the spinal cord is one mechanism underlying the recovery of function after hemisection.