Growth-associated Phosphoprotein Research Articles

Promotion of axon regeneration and inhibition of astrocyte activation by alpha A-crystallin on crushed optic nerve.

To explore the effects of αA-crystallin in astrocyte gliosis after optic nerve crush (ONC) and the mechanism of α-crystallin in neuroprotection and axon regeneration. ONC was established on the Sprague-Dawley rat model and αA-crystallin (10(-4) g/L, 4 µL) was intravitreously injected into the rat model. Flash-visual evoked potential (F-VEP) was examined 14d after ONC, and the glial fibrillary acidic protein (GFAP) levels in the retina and crush site were analyzed 1, 3, 5, 7 and 14d after ONC by immunohistochemistry (IHC) and Western blot respectively. The levels of beta Tubulin (TUJ1), growth-associated membrane phosphoprotein-43 (GAP-43), chondroitin sulfate proteoglycans (CSPGs) and neurocan were also determined by IHC 14d after ONC. GFAP level in the retina and the optic nerve significantly increased 1d after ONC, and reached the peak level 7d post-ONC. Injection of αA-crystallin significantly decreased GFAP level in both the retina and the crush site 3d after ONC, and induced astrocytes architecture remodeling at the crush site. Quantification of retinal ganglion cell (RGC) axons indicated αA-crystallin markedly promoted axon regeneration in ONC rats and enhanced the regenerated axons penetrated into the glial scar. CSPGs and neurocan expression also decreased 14d after αA-crystallin injection. The amplitude (N1-P1) and latency (P1) of F-VEP were also restored. Our results suggest α-crystallin promotes the axon regeneration of RGCs and suppresses the activation of astrocytes.

ISSLS Prize Winner

Animal model of intravertebral disc (IVD) degeneration. To examine production of inflammatory mediators in IVDs and neuropeptides in dorsal root ganglia (DRGs) in rat models of IVD compression and injury. Sensory nerve fibers in IVDs and inflammatory mediator responses have been verified in animal models of IVD injury. However, the IVD injury in animals incompletely models degenerated human IVDs causing discogenic low back pain, because human IVDs are also subject to compression. Experimental groups (controls, IVD injury, IVD compression, and their combination) of Sprague Dawley rats were prepared. Fluoro-Gold (FG; Fluorochrome, Denver, CO) was applied into coccygeal IVDs. Inflammatory mediators in IVDs, including nerve growth factor, tumor necrosis factor α, interleukin 1β, and interleukin 6, were quantified using enzyme-linked immunosorbent assays. DRGs were immunostained for calcitonin gene-related peptide, activating transcription factor 3, and growth-associated phosphoprotein 43. The upregulation of inflammatory mediators was transient in the IVD injury group but delayed and long-lasting in the IVD compression group. When the IVD injury and compression were combined, the upregulation of inflammatory mediators was long-lasting through 8 weeks. The proportion of calcitonin gene-related peptide-immunoreactive neurons among Fluoro-Gold-labeled neurons remained significantly higher in the IVD injury, compression, and combination groups than in the controls. In contrast, increases in the proportions of activating transcription factor 3-immunoreactive or growth-associated phosphoprotein 43-immunoreactive neurons in the IVD injury group animals were transient but long-lasting in the compression and combination groups compared with controls. Disc injury in rats produces persistent increases in neuropeptides in DRGs but only transient increases in inflammatory mediators in IVDs. On the contrary, disc compression in rats produces a long-lasting increase in inflammatory mediators in IVDs and neuropeptides in DRGs. Moreover, disc compression induces persistent nerve injury and regeneration of the afferent fibers innervating IVDs.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma.First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-beta. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.

Extracorporeal shockwaves induce the expression of ATF3 and GAP-43 in rat dorsal root ganglion neurons

Although extracorporeal shockwave has been applied in the treatment of various diseases, the biological basis for its analgesic effect remains unclear. Therefore, we investigated the dorsal root ganglion neurons of rats following shockwave exposure to the footpad to elucidate its effect on the peripheral nervous system. We used activating transcription factor 3 (ATF3) and growth-associated phosphoprotein (GAP-43) as markers for nerve injury and axonal regeneration, respectively. The average number of neurons immunoreactive for ATF3 increased significantly in the treated rats at all experimental time points, with 78.3% of those neurons also exhibiting immunoreactivity for GAP-43. Shockwave exposure induced injury of the sensory nerve fibers within the exposed area. This phenomenon may be linked to the desensitization of the exposure area, not the cause of pain, considering clinical research with a particular absence of painful adverse effect. Subsequent active axonal regeneration may account for the reinnervation of exposed area and the amelioration of the desensitization.

Cell proliferation and growth-associated protein 43 expression in the olfactory epithelium in Poecilia reticulata after copper solution exposure.

This study investigated the regeneration in the olfactory mucosa of the teleostean fish Poecilia reticulata when returned to dechlorinated tap water after 4-day exposure to 30 microg/L of Cu(2+). The regeneration process in the olfactory tissue was examined in fishes at 0, 3, 6 and 10 days of recovery in well water. Jade B staining permitted to evaluate the rate of the damage which was especially extended to olfactory neurons. Immediately after the end of exposure, a massive mitotic activity in the basal region of the mucosa was detected by immunostaining with PCNA. After 3 days of recovery the nuclei of the newly formed cells had already finished their migration to the upper portion of the epithelium, and cellular division was much less intense. Simultaneously, immunoreactivity for the neural growth-associated phosphoprotein GAP-43 increased respect to control levels, revealing that the new differentiating PCNA-positive elements belonged to immature neurons. After 6 days in well water no mitotic activity was detected, while the GAP-43 labelling appeared particularly concentrated in the apical surface of the olfactory epithelium. After 10 days the aspect of the olfactory epithelium was almost identical to the control. The present results suggest that after 10 days regeneration seems to be complete and integrity of the tissue restored. Furthermore, the epithelium reconstitution does not show apparent divergence from other fishes or mammals.

Recovery of the olfactory receptor neurons in the African Tilapia mariae following exposure to low copper level

Low levels of Cu 2+ are known to specifically cause olfactory neuron death in fish olfactory epithelium. This study investigated the morphological changes in the olfactory mucosa of the cichlid Tilapia mariae, after a 4-day exposure to different concentrations of Cu 2+ (20, 40 and 100 μg/l), and the regeneration time-frame, when fish exposed to 20 μg/l were returned to dechlorinated tap water. Light microscopy, combined with Fluoro Jade-B staining, permitted the observation of a dose-dependent damage which became less severe and more circumscribed to receptor cells when Cu 2+ concentration decreased. The regeneration process in the olfactory tissue was examined in fish after 0, 3, and 10 days of recovery in well water. Immunostaining with PCNA showed a massive mitotic activity in the basal region of the mucosa immediately after exposure was terminated. The mitotically produced elements were immature neurons since they expressed the neural growth-associated phosphoprotein GAP-43. After 3 days of recovery the nuclei had already completed their migration to the upper portion of the epithelium and mitotic activity was much less intensive. After 10 days the olfactory tissue did not present differences when compared to the control tissue. These results suggest that after 10 days the regeneration is completed and the integrity of the tissue restored.

Apnea, glial apoptosis and neuronal plasticity in the arousal pathway of victims of SIDS

Of 27,000 infants whose sleep-wake characteristics were studied under the age of 6 months, 38 died unexpectedly 2–12 weeks after the sleep recording in a pediatric sleep laboratory. Of these infants, 26 died of sudden infant death syndrome (SIDS), and 12 of definitely identified causes. The frequency and duration of sleep apneas were analysed. Sleep recordings and brainstem histopathology were studied to elucidate the possible relationship between sleep apnea and neuropathological changes within the arousal system. Immunohistochemical analyses were conducted using tryptophan hydroxylase (TrypH), a serotonin synthesizing enzyme, and growth-associated phosphoprotein 43 (GAP43), a marker of synaptic plasticity. The terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was used for apoptosis. The pathological and physiological data were correlated for each infant. In the SIDS victims, statistically significant positive correlations were seen between the number of TrypH-positive neurons in the dorsal raphe nucleus of the midbrain and the duration of central apneas ( p = 0.03), between the number of TUNEL-positive glial cells in the pedunculopontine tegmental nucleus (PPTN) and the average number of spines in GAP43-positive neurons in the PPTN ( p = 0.04). These findings in the dorsal raphe nucleus of the midbrain and PPTN, that play important roles in the arousal pathway suggest a possible link between changes in arousal and SIDS.

The relationship between neuronal plasticity and serotonergic neurons in the brainstem of SIDS victims

Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. Recently, the medullary serotonergic network deficiency theory has been proposed and an association between SIDS and neuronal plasticity has also been suggested. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and is critical for normal development of the serotonergic innervation. Therefore, the characteristics of GAP43-positive elements and their association with serotonergic neurons were here investigated in the brainstem of SIDS victims. Materials and methods: The materials of this study included 26 cases of SIDS and 12 control cases. The brainstem material was collected and the immunohistochemistry of GAP43 and tryptophan hydroxylase (TrypH) carried out. The density of GAP43-positive neurons and dendrites and of TrypH-positive neurons were measured quantitatively. Non-parametric analyses of GAP43 between SIDS and non-SIDS and correlation analyses between GAP43 and TrypH were performed. Results: No significant difference in GAP43-associated findings was found between SIDS and non-SIDS nor any significant correlation between GAP43-associated findings and TrypH-positive neurons. Conclusions: The results of this study were not in agreement with the association of GAP43 with SIDS and with serotonergic innervation in SIDS.

The relationship between neuronal plasticity and serotonergic neurons in the brainstem of SIDS victims

Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. Recently, the medullary serotonergic network deficiency theory has been proposed and an association between SIDS and neuronal plasticity has also been suggested. The growth-associated phosphoprotein 43 (GAP43) is a marker of synaptic plasticity and is critical for normal development of the serotonergic innervation. Therefore, the characteristics of GAP43-positive elements and their association with serotonergic neurons were here investigated in the brainstem of SIDS victims. Materials and methods: The materials of this study included 26 cases of SIDS and 12 control cases. The brainstem material was collected and the immunohistochemistry of GAP43 and tryptophan hydroxylase (TrypH) carried out. The density of GAP43-positive neurons and dendrites and of TrypH-positive neurons were measured quntitatively. Nonparametric analyses of GAP43 between SIDS and non-SIDS and correlation analyses between GAP43 and TrypH were performed. Results: No significant difference in GAP43-associated findings was found between SIDS and non-SIDS nor any significant correlation between GAP43-associated findings and TrypH-positive neurons. Conclusions:. The results of this study were not in agreement with the association of GAP43 with SIDS and with serotonergic innervation in SIDS.

Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury

An experimental study was performed to investigate the influence of the inflammation in peripheral target tissue on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia (DRG) and on recovery of crushed nerve. Fifty-four male Wistar rats were used for this study. The sciatic nerve was operatively crushed unilaterally with an aneurysm clip. Inflammation in peripheral target tissue was induced by injection of complete Freund's adjuvant (CFA) at 1 week before crush. In crushed or crushed with arthritis rats DRGs were examined in immunohistochemistry for GAP-43 and the sciatic nerves were observed in Epon embedded sections with toluidine blue stain. In addition, electrophysiological studies of the nerves were performed to evaluate the recovery of function. Immunohistochemical studies showed the ratio of GAP-43 immunopositive cells in crushed with arthritis rats was significantly lower than that in crushed rats at 1 week after crush ( P<0.01). Electrophysiological studies at 4 weeks after crush showed functional nerve recovery in crushed with arthritis rats was significantly suppressed compared with that in crushed rats ( P<0.01). Histological studies showed the mean diameter of the axons in crushed with arthritis rats was significantly smaller than that in crushed rats ( P<0.01). All these findings indicate that inflammation in peripheral target tissue suppresses GAP-43 expression in DRG and eventually suppresses functional and morphological recovery of the crushed nerve.

Arousal deficiency theory in sudden infant death syndrome with reference to neuronal plasticity

Objective: Among 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age (including 26 infant victims of sudden infant death syndrome (SIDS), five with congenital cardiac abnormalities, two from infected pulmonary dysplasia, two from septic shock with multi-organ failure, one with a prolonged seizure, one from prolonged neonatal hypoxemia and one from meningitis and brain infarction). Method: The frequency and duration of sleep apnea events recorded some 3–12 weeks before the infants' deaths were analyzed. Brainstem material from these 38 infants was studied in an attempt to elucidate the relationship between sleep apnea and neuronal pathological changes in the arousal pathway. The histochemical analyses included Bielschowsky staining and the immunohistochemical analyses included the evaluation of growth-associated phosphoprotein 43 (GAP43) and of synaptophysin as markers for synaptic plasticity. Neurofibrae with positive pathological reactions were quantitatively analyzed. Pathological and physiological data were linked for each infant. Results: The correlation between sleep apnea and neuronal plasticity in the arousal pathway of the SIDS victims was not seen in the control infants and the correlation between sleep apnea and neuronal plasticity in the arousal pathway found in the control infants was not seen in the SIDS victims. Conclusion: These findings suggest that neuronal plasticity in the brainstem arousal pathway is related with SIDS.

Desmoplastic and spindle cell melanomas express protein markers of the neural crest but not of later committed stages of Schwann cell differentiation.

The rare desmoplastic and spindle cell variants of malignant melanoma exhibit histological and biochemical features suggestive of early Schwann cell differentiation. These features include a spindle-shaped morphology, neurotropism, and the expression of the low affinity nerve growth factor receptor (p75NGFR). We evaluated by immunohistochemistry (using formalin-fixed, paraffin-embedded tissues) nine desmoplastic and three spindle cell melanomas for the expression of peripherin, p75NGFR, neural cell adhesion molecule (CD56/N-CAM), and growth-associated phosphoprotein-43 (GAP-43). Peripherin is expressed in the neural crest and in neurons, but not in cells committed to the Schwann cell lineage. p75NGFR and CD56/N-CAM also are expressed in early neural crest cells, but persist in unmyelinated and early premyelinating Schwann cells. GAP-43 is expressed in unmyelinated Schwann cells, but is downregulated in the later premyelinating to promyelinating stages of cells committed to the Schwann cell lineage. Peripherin was expressed in 7/12 (58%), p75NGFR in 4/12 (33%), and CD56/N-CAM in 6/12 (50%) of the desmoplastic and spindle cell melanomas. GAP-43 was not expressed (0%) in any of the 12 melanomas (chi2, p = 0.05). Desmoplastic and spindle cell melanomas express protein markers common to cells of the neural crest and to neurons similar to the immunophenotype previously reported for epithelioid cell melanomas. The expression of peripherin and the lack of expression of GAP-43 further define that these rare subtypes of melanoma do not recapitulate the later committed stages of Schwann cell differentiation.

From physiology to pathology: arousal deficiency theory in sudden infant death syndrome (SIDS)—with reference to apoptosis and neuronal plasticity

Among 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age (including 26 infant victims of sudden infant death syndrome (SIDS), 5 with congenital cardiac abnormalities, 2 from infected pulmonary dysplasia, 2 from septic shock with multi-organ failure, 1 with a prolonged seizure, 1 from prolonged neonatal hypoxemia, 1 from meningitis and brain infarction). The frequency and duration of sleep apneas recorded some 3–12 weeks before the infants’ death were analyzed. Brainstem material from these 38 infants was studied in an attempt to elucidate the relationship between sleep apnea and neuronal pathological changes in the arousal pathway. Immunohistochemical analyses included the evaluation of growth-associated phosphoprotein 43 (GAP43) as a marker for synaptic plasticity. The terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was used to identify apoptosis. The positive pathological reactions were quantitatively analyzed. The pathological and physiological data were linked for each infant. Akaike Information Criterion (AIC) statistics was calculated to elucidate the relationship between the physiological and the pathological data in the SIDS victims. The findings illustrated the possibility of an organic fragility within the arousal pathway, particularly in the midbrain periaqueductal gray matter, which is associated with the “visceral alerting response”. This autonomic response occurs within an acetylcholine afferent system and pedunculopontine tegmental nucleus (PPTN). The finding is, in future SIDS infants, associated with repetitive sleep apnea.

Divergent Cellular Differentiation Pathways during the Invasive Stage of Cutaneous Malignant Melanoma Progression

Melanocytic nevus cells in the dermis adopt many morphological features of Schwann cells. These differentiation-related changes typically are not observed in melanomas. However, nevus cells do not fully recapitulate a Schwann cell phenotype, because they lack expression of mature myelin-associated proteins. In this study, melanocytic nevi and malignant melanomas were examined by immunohistochemistry for expression of low-affinity nerve growth factor receptor (p75NGFR), neural cell adhesion molecule (CD56/N-CAM), and growth-associated phosphoprotein-43 (GAP-43). These three proteins define the earliest stages of Schwann cell development but are not expressed in myelinated Schwann cells or normal melanocytes. p75NGFR was expressed in 25 of 25 (100%) and CD56/N-CAM and GAP-43 in 23 of 25 (92%) nevi, predominantly in type C nevus cells and nevic corpuscles. Most (84%) of the nevi expressed all three proteins. In primary invasive and metastatic melanoma, expression of each of the three proteins was limited to </=20% of lesions but was not observed in any melanoma in situ (chi(2 )P < 0.0001). None of the melanomas expressed all three proteins (ANOVA P < 0.0001). These data confirm and extend earlier studies by showing that terminal differentiation of melanocytes in the dermis recapitulates some aspects observed in the earliest stages of Schwann cell development and that invasive melanomas follow a divergent pathway. Studying these early differentiation events may help to identify specific defects in the relevant signaling pathways and establish tenable targets for therapy of advanced-stage melanoma.

Growth-associated phosphoprotein expression is increased in the supragranular regions of the dentate gyrus following pilocarpine-induced seizures in rats

Neuroplasticity has been investigated considering the neuronal growth-associated phosphoprotein as a marker of neuronal adaptive capabilities. In the present work, studying the hippocampal reorganization observed in the epilepsy model induced by pilocarpine, we carried out quantitative western blotting associated with immunohistochemistry to determine the distribution of growth-associated phosphoprotein in the hippocampus of rats in acute, silent and chronic periods of this epilepsy model. The fibers and punctate elements from the inner molecular layer of the dentate gyrus were strongly immunostained in animals killed 5 h after status epilepticus, compared with the same region in control animals. Rats presenting partial seizures showed no alterations in the immunostaining pattern compared with saline-treated animals. The hippocampal dentate gyrus of animals during the seizure-free period and presenting spontaneous recurrent seizures was also characterized by strong growth-associated phosphoprotein immunostaining of fibers and punctate elements in the inner molecular layer, contrasting with the control group. As determined by western blotting analysis, growth-associated phosphoprotein levels increased following status epilepticus and remained elevated at the later time-points, both during the silent period and during the period of chronic recurring seizures. Pilocarpine-treated animals, which did not develop status epilepticus, showed no change in growth-associated phosphoprotein levels, indicating that status epilepticus is important to induce growth-associated phosphoprotein overexpression. The measurement of this overexpression could represent one of the early signals of hippocampal reorganization due to status epilepticus-induced damage.

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury

A possible role for nitric oxide in growth and regeneration of dorsal root ganglion (DRG) afferents has been explored in lesion experiments by comparing immunocytochemistry for nitric oxide synthase (NOS) with that for the growth-associated phosphoprotein 43 (GAP-43). Sciatic nerve ligature induced a progressive increase in the number of small DRG cell profiles immunopositive for NOS between 2 days and 4 weeks of survival. In the proximal stump of the ligature, NOS-immunopositive fibers began to appear 2 days after injury and their growth cones were especially evident after 7 days. NOS-immunopositive fibers appeared past (i.e., distal to) the ligature at 14 days of survival and extended for at least 6 mm in either direction 4 weeks after the lesion. Dorsal root ligature alone at L4-L5 did not result in expression of NOS in DRG neurons or in the appearance of NOS-immunopositive fibers. In rats with dorsal root ligature and nerve ligature, the results were similar to those with nerve ligature only. DRG cell profiles immunopositive for GAP-43 kept increasing from 2 days to 4 weeks after sciatic nerve ligature and included small neurons initially and large neurons subsequently. Numerous axons became GAP-43 immunopositive on both sides of the ligature from 2 days after injury. In double-labeled material, about 80% of DRG cell profiles immunopositive for NOS were also immunopositive for GAP-43. The two antigens co-occurred in peripheral nerve axons proximal to the ligature starting at about 7 days and distal to it at about 2 weeks after ligature. Thus, in response to nerve lesion, nitric oxide may not only provide an injury signal to the central nervous system but may also contribute to the growth and regeneration of injured axons.

MAP2 and GAP-43 expression in normal and weaver mouse cerebellum: correlative immunohistochemical and in situ hybridization studies.

MAP2 is a major microtubule-associated brain protein, selectively localized in dendrites; growth-associated phosphoprotein GAP-43 is a neuron-specific protein associated with axonal outgrowth. In adult cerebellum, both of these proteins and their corresponding RNA transcripts are most strongly expressed by granule cells. Using immunocytochemistry with antibodies and in situ hybridization histochemistry with [32P] labeled oligonucleotide probes, we examined the cellular localization of MAP2, GAP-43 and their mRNAs in the cerebellum of control and weaver (wv/wv) mutant mice, which exhibit massive granule cell death. In wild-type (+/+) mice, MAP2 immunoreactivity was seen in neuronal somata and dendrites of the granule cell layer; GAP-43 immunoreactivity was present in molecular layer, corresponding to the distribution of parallel fibres. Transcripts encoding MAP2 and GAP-43 were localized in the layer of the granule cell somata. In heterozygous weaver mice (wv/+), which feature an intermediate degree of granule cell loss, MAP2 immunoreactivity was localized in the granular layer, and the pattern of GAP-43 immunostaining was also similar to +/+, the only difference being a thinner molecular layer. Heterozygotes had an anatomical pattern of MAP2 and GAP-43 mRNA hybridization qualitatively similar to that of the wild-type with some deviations in signal intensity. In homozygous weaver mutants (wv/wv), MAP2 immunoreactivity was extremely weak in the area beneath Purkinje cells and a certain GAP-43 immunoreactivity was seen in the upper part of cerebellar cortex. Hybridization signals for MAP2 and GAP-43 mRNAs were minimal. The reported alterations in regional pattern of MAP2 and GAP-43 expression in mutant mice offer a molecular correlate of dendritic and axonal protein gene transcription pertinent to the neuropathological manifestations of certain forms of heredodegenerative ataxia.

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phopshatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.

Presynaptic phosphoprotein B-50/GAP-43 in neuronal and synaptic plasticity.

B-50/GAP-43 is a growth-associated phosphoprotein enriched in growth cones and in the presynaptic terminal. The expression of the protein is restricted to the nervous system and is highest in the first week after birth. In adult brain, B-50 is enriched in areas with high plasticity. The regulation of expression of the B-50 gene occurs both at the transcriptional and post-transcriptional level by unknown mechanisms. The gene contains 2 regions displaying promoter activity, the most 3' of which (P2) is the active on in vivo. Expression of B-50 in non-neuronal cells results in filopodial extensions whereas antibodies or antisense oligo's to B-50 prevent neurite outgrowth. The protein is important for neuronal pathfinding. Several post-translational modifications have been described, ADP-ribosylation and palmitoylation in the membrane binding domain, phosphorylation by PKC, casein kinase II and phosphorylase kinase, and dephosphorylation by several phosphatases, among which is calcineurin. Interactions of B-50 have been described with calmodulin, PIP kinase, F-actin, and phospholipids. Recent studies indicate that the phosphorylation state and amount of calmodulin bound to B-50 regulate the rate of transmitter release. Induction of long-term potentiation by high frequency stimulation of hippocampal slices results in an increased state of B-50 phosphorylation. This will increase the amount of free calmodulin in the presynaptic terminal and increase the amount of transmitter released. Although B-50 is involved in seemingly unrelated forms of neuronal plasticity, neurite outgrowth and transmitter release, our unifying hypothesis is that the protein plays an (unknown) essential, modulatory role in membrane expansion.