Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, Fe, Ht||IHC, IP, WB||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at 2-8°C in undiluted for up to 6 months after date of receipt.|
|Material Size||50 µg|
References | 62 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Gap43 transcription modulation in the adult brain depends on sensory activity and synaptic cooperation. |
Rosskothen-Kuhl, N; Illing, RB
PloS one 9 e92624 2014
Brain development and learning is accompanied by morphological and molecular changes in neurons. The growth associated protein 43 (Gap43), indicator of neurite elongation and synapse formation, is highly expressed during early stages of development. Upon maturation of the brain, Gap43 is down-regulated by most neurons with the exception of subdivisions such as the CA3 region of hippocampus, the lateral superior olive (LSO) and the central inferior colliculus (CIC). Little is known about the regulation of this mRNA in adult brains. We found that the expression of Gap43 mRNA in specific neurons can be modulated by changing sensory activity of the adult brain. Using the central auditory system of rats as a model, Gap43 protein and mRNA levels were determined in LSO and CIC of hearing-experienced rats unilaterally or bilaterally deafened or unilaterally stimulated by a cochlear implant (CI). Our data indicate that Gap43 is a marker useful beyond monitoring neuronal growth and synaptogenesis, reflecting also specific patterns of synaptic activities on specific neurons. Thus, unilateral loss of input to an adult auditory system directly causes asymmetrical expression of Gap43 mRNA between LSOs or CICs on both sides of the brainstem. This consequence can be prevented by simple-patterned stimulation of a dysfunctional ear by way of a CI. We suggest that as a function of input balance and activity pattern, Gap43 mRNA expression changes as cells associate converging afferent signals.
|Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina. |
de Andrade, GB; Kunzelman, L; Merrill, MM; Fuerst, PG
Molecular vision 20 1422-33 2014
The Down syndrome cell adhesion molecule (Dscam) gene is required for normal dendrite arborization and lamination in the mouse retina. In this study, we characterized the developmental localization of the DSCAM protein to better understand the postnatal stages of retinal development during which laminar disorganization occur in the absence of the protein.Immunohistochemistry and colocalization analysis software were used to assay the localization of the DSCAM protein during development of the retina.We found that DSCAM was initially localized diffusely throughout mouse retinal neurites but then adopted a punctate distribution. DSCAM colocalized with catenins in the adult retina but was not detected at the active zone of chemical synapses, electrical synapses, and tight junctions. Further analysis identified a wave of colocalization between DSCAM and numerous synaptic and junction proteins coinciding with synaptogenesis between bipolar and retinal ganglion cells.Research presented in this study expands our understanding of DSCAM function by characterizing its location during the development of the retina and identifies temporally regulated localization patterns as an important consideration in understanding the function of adhesion molecules in neural development.
|Up-regulation of GAP-43 in the chinchilla ventral cochlear nucleus after carboplatin-induced hearing loss: correlations with inner hair cell loss and outer hair cell loss. |
Kraus, KS; Ding, D; Jiang, H; Kermany, MH; Mitra, S; Salvi, RJ
Hearing research 302 74-82 2013
Inner ear damage leads to nerve fiber growth and synaptogenesis in the ventral cochlear nucleus (VCN). In this study, we documented the relationship between hair cell loss patterns and synaptic plasticity in the chinchilla VCN using immunolabeling of the growth associated protein-43 (GAP-43), a protein associated with axon outgrowth and modification of presynaptic endings. Unilateral round window application of carboplatin caused hair cell degeneration in which inner hair cells (IHC) were more vulnerable than outer hair cells (OHC). One month after carboplatin treatment (0.5-5 mg/ml), we observed varying patterns of cochlear hair cell loss and GAP-43 expression in VCN. Both IHC loss and OHC loss were strongly correlated with increased GAP-43 immunolabeling in the ipsilateral VCN. We speculate that two factors might promote the expression of GAP-43 in the VCN; one is the loss of afferent input through IHC or the associated type I auditory nerve fibers. The other occurs when the medial olivocochlear efferent neurons lose their cochlear targets, the OHC, and may as compensation increase their synapse numbers in the VCN.
|Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat. |
Amemori, T; Romanyuk, N; Jendelova, P; Herynek, V; Turnovcova, K; Prochazka, P; Kapcalova, M; Cocks, G; Price, J; Sykova, E
Stem cell research & therapy 4 68 2013
A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI).A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes.Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)(+) axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2(+) and choline acetyltransferase (ChAT)(+) neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2.The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.
|Chondroitinase enhances cortical map plasticity and increases functionally active sprouting axons after brain injury. |
Harris, NG; Nogueira, MS; Verley, DR; Sutton, RL
Journal of neurotrauma 30 1257-69 2013
The beneficial effect of interventions with chondroitinase ABC enzyme to reduce axon growth-inhibitory chondroitin sulphate side chains after central nervous system injuries has been mainly attributed to enhanced axonal sprouting. After traumatic brain injury (TBI), it is unknown whether newly sprouting axons that occur as a result of interventional strategies are able to functionally contribute to existing circuitry, and it is uncertain whether maladaptive sprouting occurs to increase the well-known risk for seizure activity after TBI. Here, we show that after a controlled cortical impact injury in rats, chondroitinase infusion into injured cortex at 30 min and 3 days reduced c-Fos⁺ cell staining resulting from the injury alone at 1 week postinjury, indicating that at baseline, abnormal spontaneous activity is likely to be reduced, not increased, with this type of intervention. c-Fos⁺ cell staining elicited by neural activity from stimulation of the affected forelimb 1 week after injury was significantly enhanced by chondroitinase, indicating a widespread effect on cortical map plasticity. Underlying this map plasticity was a larger contribution of neuronal, rather than glial cells and an absence of c-Fos⁺ cells surrounded by perineuronal nets that were normally present in stimulated naïve rats. After injury, chondroitin sulfate proteoglycan digestion produced the expected increase in growth-associated protein 43-positive axons and perikarya, of which a significantly greater number were double labeled for c-Fos after intervention with chondroitinase, compared to vehicle. These data indicate that chondroitinase produces significant gains in cortical map plasticity after TBI, and that either axonal sprouting and/or changes in perineuronal nets may underlie this effect. Chondroitinase dampens, rather than increases nonspecific c-Fos activity after brain injury, and induction of axonal sprouting is not maladaptive because greater numbers are functionally active and provide a significant contribution to forelimb circuitry after brain injury.
|Proteoglycan abnormalities in olfactory epithelium tissue from subjects diagnosed with schizophrenia. |
Pantazopoulos, H; Boyer-Boiteau, A; Holbrook, EH; Jang, W; Hahn, CG; Arnold, SE; Berretta, S
Schizophrenia research 150 366-72 2013
Emerging evidence points to proteoglycan abnormalities in the pathophysiology of schizophrenia (SZ). In particular, markedly abnormal expression of chondroitin sulfate proteoglycans (CSPGs), key components of the extracellular matrix, was observed in the medial temporal lobe. CSPG functions, including regulation of neuronal differentiation and migration, are highly relevant to the pathophysiology of SZ. CSPGs may exert similar functions in the olfactory epithelium (OE), a continuously regenerating neural tissue that shows cell and molecular abnormalities in SZ. We tested the hypothesis that CSPG expression in OE may be altered in SZ. CSPG-positive cells in postmortem OE from non-psychiatric control (n=9) and SZ (n=10) subjects were counted using computer-assisted light microscopy. 'Cytoplasmic' CSPG (c-CSPG) labeling was detected in sustentacular cells and some olfactory receptor neurons (c-CSPG+ORNs), while 'pericellular' CSPG (p-CSPG) labeling was found in basal cells and some ORNs (p-CSPG+ORNs). Dual labeling for CSPG and markers for mature and immature ORNs suggests that c-CSPG+ORNs correspond to mature ORNs, and p-CSPG+ORNs to immature ORNs. Previous studies in the same cohort demonstrated that densities of mature ORNs were unaltered (Arnold et al., 2001). In the present study, numerical densities of c-CSPG+ORNs were significantly decreased in SZ (pless than 0.025; 99.32% decrease), suggesting a reduction of CSPG expression in mature ORNs. Previous studies showed a striking increase in the ratios of immature neurons with respect to basal cells. In this study, we find that the ratio of p-CSPG+ORNs/CSPG+basal cells was significantly increased (p=0.03) in SZ, while numerical density changes of p-CSPG+ORNs (110.71% increase) or CSPG+basal cells (53.71% decrease), did not reach statistical significance. Together, these results indicate that CSPG abnormalities are present in the OE of SZ and specifically point to a reduction of CSPG expression in mature ORNs in SZ. Given the role CSPGs play in OE cell differentiation and axon guidance, we suggest that altered CSPG expression may contribute to ORN lineage dysregulation, and olfactory identification abnormalities, observed in SZ.
|DISC1 variants 37W and 607F disrupt its nuclear targeting and regulatory role in ATF4-mediated transcription. |
Malavasi, EL; Ogawa, F; Porteous, DJ; Millar, JK
Human molecular genetics 21 2779-92 2012
Disrupted-In-Schizophrenia 1 (DISC1), a strong genetic candidate for psychiatric illness, encodes a multicompartmentalized molecular scaffold that regulates interacting proteins with key roles in neurodevelopment and plasticity. Missense DISC1 variants are associated with the risk of mental illness and with brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. We examined the effect of rare and common DISC1 amino acid substitutions on subcellular targeting. We report that both the rare putatively causal variant 37W and the common variant 607F independently disrupt DISC1 nuclear targeting in a dominant-negative fashion, predicting that DISC1 nuclear expression is impaired in 37W and 607F carriers. In the nucleus, DISC1 interacts with the transcription factor Activating Transcription Factor 4 (ATF4), which is involved in the regulation of cellular stress responses, emotional behaviour and memory consolidation. At basal cAMP levels, wild-type DISC1 inhibits the transcriptional activity of ATF4, an effect that is weakened by both 37W and 607F independently, most likely as a consequence of their defective nuclear targeting. The common variant 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. We also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is ablated by 37W and 607F. By showing that DISC1 amino acid substitutions associated with psychiatric illness affect its regulatory function in ATF4-mediated transcription, our study highlights a potential mechanism by which these variants may impact on transcriptional events mediating cognition, emotional reactivity and stress responses, all processes of direct relevance to psychiatric illness.
|Differential effects of AdOx on gene expression in P19 embryonal carcinoma cells. |
Yan, L; Zhao, HY; Zhang, Y; Shen, YF
BMC neuroscience 13 6 2012
Pluripotent cells maintain a unique gene expression pattern and specific chromatin signature. In this study, we explored the effect of the methyltransferase inhibitor adenosine dialdehyde (AdOx) on pluripotency maintenance and gene expression in P19 embryonal carcinoma cells.After AdOx treatment, the pluripotency-related gene network became disordered, and the early developmental genes were released from the repression. Remarkably, AdOx caused contrasting effects on the expression of two key pluripotency genes, nanog and oct3/4, with the reduction of the repressive histone marks H3K27me3, H3K9me3 and H3K9me2 only in the nanog gene.Key pluripotency genes were controlled by different mechanisms, including the differential enrichment of repressive histone methylation marks. These data provided novel clues regarding the critical role of histone methylation in the maintenance of pluripotency and the determination of cell fate in P19 pluripotent cells.
|The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response. |
Stephan, AB; Tobochnik, S; Dibattista, M; Wall, CM; Reisert, J; Zhao, H
Nature neuroscience 15 131-7 2012
Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception.
|Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice. |
Cygnar, KD; Collins, SE; Ferguson, CH; Bodkin-Clarke, C; Zhao, H
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 14557-62 2012
Feedback inhibition of adenylyl cyclase III (ACIII) via Ca(2+)-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine(1076) of ACIII, the only residue responsible for Ca(2+)-induced phosphorylation and inhibition of ACIII (Wei et al., 1996, 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wild-type and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine(1076) plays a far less important role in olfactory response attenuation than previously thought.
|Pigment epithelium-derived factor associates with neuropathy and fibrosis in pancreatic cancer. |
Samkharadze T, Erkan M, Reiser-Erkan C, Demir IE, Kong B, Ceyhan GO, Michalski CW, Esposito I, Friess H, Kleeff J.
The American journal of gastroenterology 106 968-80 2011
Pigment epithelium-derived factor (PEDF) is a noninhibitory member of the serine protease inhibitor gene family with neuroprotective, neuroproliferative, and anti-angiogenic functions. Its role in pancreatic fibrosis and neuropathy is unknown.
|Endogenous prolactin generated during peripheral inflammation contributes to thermal hyperalgesia. |
Scotland, PE; Patil, M; Belugin, S; Henry, MA; Goffin, V; Hargreaves, KM; Akopian, AN
The European journal of neuroscience 34 745-54 2011
Prolactin (PRL) is a hormone and a neuromodulator. It sensitizes TRPV1 (transient receptor potential cation channel subfamily V member 1) responses in sensory neurons, but it is not clear whether peripheral inflammation results in the release of endogenous PRL, or whether endogenous PRL is capable of acting as an inflammatory mediator in a sex-dependent manner. To address these questions, we examined inflammation-induced release of endogenous PRL, and its regulation of thermal hyperalgesia in female and male rats. PRL is expressed in several types of peripheral neuronal and non-neuronal cells, including TRPV1-positive nerve fibers, preadipocytes and activated macrophages/monocytes localized in the vicinity of nerves. Evaluation of PRL levels in hindpaws and plasma indicated that complete Freund's adjuvant (CFA) stimulates release of peripheral, but not systemic, PRL within 6-48 h in both ovariectomized females with estradiol replacement (OVX-E) and intact male rats. The time course of release varies in OVX-E and intact male rats. We next employed the prolactin receptor (PRL-R) antagonist Δ1-9-G129R-hPRL to assess the role of locally produced PRL in nociception. Applied at a ratio of 1 : 1 (PRL:Δ1-9-G129R-hPRL; 40 nm each), this antagonist was able to nearly (≈ 80%) reverse PRL-induced sensitization of capsaicin responses in rat sensory neurons. CFA-induced inflammatory thermal hyperalgesia in OVX-E rat hindpaws was significantly reduced in a dose-dependent manner by the PRL-R antagonist at 6 h but not at 24 h. In contrast, PRL contributed to inflammatory thermal hyperalgesia in intact male rats at 24, but not at 6 h. These findings indicate that inflammation leads to accumulation of endogenous PRL in female and male rats. Furthermore, PRL acts as an inflammatory mediator at different time points for female and intact male rats.
|Deafferentation-induced redistribution of MMP-2, but not of MMP-9, depends on the emergence of GAP-43 positive axons in the adult rat cochlear nucleus. |
Fredrich, M; Illing, RB
Neural plasticity 2011 859359 2011
The matrix metalloproteinases MMP-9 and MMP-2, major modulators of the extracellular matrix (ECM), were changed in amount and distribution in the rat anteroventral cochlear nucleus (AVCN) following its sensory deafferentation by cochlear ablation. To determine what causal relationships exist between the redistribution of MMP-9 and MMP-2 and deafferentation-induced reinnervation, kainic acid was stereotaxically injected into the ventral nucleus of the trapezoid body (VNTB) prior to cochlear ablation, killing cells that deliver the growth associated protein 43 (GAP-43) into AVCN. Deafferentation-induced changes in the pattern of MMP-9 staining remained unaffected by VNTB lesions. By contrast, changes in the distribution of MMP-2 normally evoked by sensory deafferentation were reversed if GAP-43 positive axons were prevented to grow in AVCN. In conclusion, GAP-43-containing axons emerging in AVCN after cochlear ablation seem to be causal for the maintenance of MMP-2-mediated ECM remodeling.
|Synaptic reorganization in the adult rat's ventral cochlear nucleus following its total sensory deafferentation. |
Hildebrandt, H; Hoffmann, NA; Illing, RB
PloS one 6 e23686 2011
Ablation of a cochlea causes total sensory deafferentation of the cochlear nucleus in the brainstem, providing a model to investigate nervous degeneration and formation of new synaptic contacts in the adult brain. In a quantitative electron microscopical study on the plasticity of the central auditory system of the Wistar rat, we first determined what fraction of the total number of synaptic contact zones (SCZs) in the anteroventral cochlear nucleus (AVCN) is attributable to primary sensory innervation and how many synapses remain after total unilateral cochlear ablation. Second, we attempted to identify the potential for a deafferentation-dependent synaptogenesis. SCZs were ultrastructurally identified before and after deafferentation in tissue treated for ethanolic phosphotungstic acid (EPTA) staining. This was combined with pre-embedding immunocytochemistry for gephyrin identifying inhibitory SCZs, the growth-associated protein GAP-43, glutamate, and choline acetyltransferase. A stereological analysis of EPTA stained sections revealed 1.11±0.09 (S.E.M.)×10(9) SCZs per mm(3) of AVCN tissue. Within 7 days of deafferentation, this number was down by 46%. Excitatory and inhibitory synapses were differentially affected on the side of deafferentation. Excitatory synapses were quickly reduced and then began to increase in number again, necessarily being complemented from sources other than cochlear neurons, while inhibitory synapses were reduced more slowly and continuously. The result was a transient rise of the relative fraction of inhibitory synapses with a decline below original levels thereafter. Synaptogenesis was inferred by the emergence of morphologically immature SCZs that were consistently associated with GAP-43 immunoreactivity. SCZs of this type were estimated to make up a fraction of close to 30% of the total synaptic population present by ten weeks after sensory deafferentation. In conclusion, there appears to be a substantial potential for network reorganization and synaptogenesis in the auditory brainstem after loss of hearing, even in the adult brain.
|Acute myocardial infarction induces bilateral stellate ganglia neural remodeling in rabbits. |
Nguyen, BL; Li, H; Fishbein, MC; Lin, SF; Gaudio, C; Chen, PS; Chen, LS
Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology 21 143-8 2011
Myocardial infarction (MI) results in cardiac nerve sprouting in the myocardium. Whether or not similar neural remodeling occurs in the stellate ganglia (SGs) is unknown. We aimed to test the hypothesis that MI induces bilateral SG nerve sprouting.Acute MI was created by coronary artery ligation in rabbits (n=12). Serum nerve growth factor (NGF) level was measured by enzyme-linked immunosorbent assay. The hearts and bilateral SGs were harvested for immunohistochemistry after 1 week in six rabbits and after 1 month in six rabbits. Immunostaining for tyrosine hydroxylase (TH), growth-associated protein 43 (GAP43), choline acetyltransferase (ChAT), and synaptophysin (SYN) was performed to determine the magnitude of nerve sprouting. Tissues from six normal rabbits were used as controls. Nerve density was determined by computerized morphometry.Myocardial infarction results in increased serum NGF levels at 1 week (1519.8±632.2 ng/ml) that persist up to 1 month (1361.2±176.3 ng/ml) as compared to controls (89.6±34.9 ng/ml) (P=.0002 and P=.0001, respectively). Immunostaining demonstrated nerve sprouting and hyperinnervation in both SGs after MI. The nerve densities (μm(2)/ganglion cell) in SG 1 week after MI and 1 month after MI and those in control groups, respectively, were as follows: GAP43: 278±96, 225±39, and 149±57 (P=.01); SYN: 244±152, 268±115, and 102±60 (P=.02); TH: 233±71, 180±50, and 135±68 (P=.047); ChAT: 244±100, 208±46, and 130±41 μm(2)/cell (P=.01).Myocardial infarction increases serum NGF levels and induces nerve sprouting and hyperinnervation in bilateral SGs for at least 1 month after MI. The hyperinnervation includes both adrenergic axons and cholinergic axons in the SG.
|Intramyocardial transplantation of human adipose-derived stromal cell and endothelial progenitor cell mixture was not superior to individual cell type transplantation in improving left ventricular function in rats with myocardial infarction. |
Hong SJ, Kihlken J, Choi SC, March KL, Lim DS
International journal of cardiology 2011
|Relationship between noise-induced hearing-loss, persistent tinnitus and growth-associated protein-43 expression in the rat cochlear nucleus: does synaptic plasticity in ventral cochlear nucleus suppress tinnitus? |
Kraus, KS; Ding, D; Jiang, H; Lobarinas, E; Sun, W; Salvi, RJ
Neuroscience 194 309-25 2011
Aberrant, lesion-induced neuroplastic changes in the auditory pathway are believed to give rise to the phantom sound of tinnitus. Noise-induced cochlear damage can induce extensive fiber growth and synaptogenesis in the cochlear nucleus, but it is currently unclear if these changes are linked to tinnitus. To address this issue, we unilaterally exposed nine rats to narrow-band noise centered at 12 kHz at 126 dB sound pressure level (SPL) for 2 h and sacrificed them 10 weeks later for evaluation of synaptic plasticity (growth-associated protein 43 [GAP-43] expression) in the cochlear nucleus. Noise-exposed rats along with three age-matched controls were screened for tinnitus-like behavior with gap prepulse inhibition of the acoustic startle (GPIAS) before, 1-10 days after, and 8-10 weeks after the noise exposure. All nine noise-exposed rats showed similar patterns of severe hair cell loss at high- and mid-frequency regions in the exposed ear. Eight of the nine showed strong up-regulation of GAP-43 in auditory nerve fibers and pronounced shrinkage of the ventral cochlear nucleus (VCN) on the noise-exposed side, and strong up-regulation of GAP-43 in the medial ventral VCN, but not in the lateral VCN or the dorsal cochlear nucleus. GAP-43 up-regulation in VCN was significantly greater in Noise-No-Tinnitus rats than in Noise-Tinnitus rats. One Noise-No-Tinnitus rat showed no up-regulation of GAP-43 in auditory nerve fibers and only little VCN shrinkage, suggesting that auditory nerve degeneration plays a role in tinnitus generation. Our results suggest that noise-induced tinnitus is suppressed by strong up-regulation of GAP-43 in the medial VCN. GAP-43 up-regulation most likely originates from medial olivocochlear neurons. Their increased excitatory input on inhibitory neurons in VCN may possibly reduce central hyperactivity and tinnitus.
|Detection of variable levels of RAR? and RAR? proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells. |
FaMitah Q Buchanan,Cecile Rochette-Egly,Mary Ann Asson-Batres
Cell and tissue research 346 2011
Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RAR? and RAR? protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RAR? antibodies and one RAR? antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RAR? antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RAR? and RAR? distribution from one cell to the other, suggesting that RAR? and RAR? protein levels were not synchronous throughout the cell population. The results indicate that RAR? and RAR? are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.
|HIV-1 Nef increases astrocyte sensitivity towards exogenous hydrogen peroxide. |
Masanetz, S; Lehmann, MH
Virology journal 8 35 2011
HIV-1 infected individuals are under chronic exposure to reactive oxygen species (ROS) considered to be instrumental in the progression of AIDS and the development of HIV-1 associated dementia (HAD). Astrocytes support neuronal function and protect them against cytotoxic substances including ROS. The protein HIV-1 Nef, a progression factor in AIDS pathology is abundantly expressed in astrocytes in patients with HAD, and thus may influence its functions.Endogenous expressed HIV-1 Nef leads to increased sensitivity of human astrocytes towards exogenous hydrogen peroxide but not towards TNF-alpha. Cell death of nef-expressing astrocytes exposed to 10 μM hydrogen peroxide for 30 min occurred within 4 h.HIV-1 Nef may contribute to neuronal dysfunction and the development of HAD by causing death of astrocytes through decreasing their tolerance for hydrogen peroxide.Full Text Article
|Enhancer of Zeste Homolog 2 Overexpression in Nasopharyngeal Carcinoma: An Independent Poor Prognosticator that Enhances Cell Growth. |
Hwang CF, Huang HY, Chen CH, Chien CY, Hsu YC, Li CF, Fang FM
Int J Radiat Oncol Biol Phys 2011
PURPOSE: As a key component of polycomb-repressive complex 2, enhancer of zeste homolog 2 (EZH2) represses target genes through histone methylation and is frequently overexpressed and associated with poor prognosis in common carcinomas. For the first time, we reported EZH2 expression and its biological and clinical significance in nasopharyngeal carcinoma (NPC).METHODS AND MATERIALS: In NPC cell lines and specimens, endogenous expression of EZH2 mRNA and protein was determined by semiquantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. To analyze the effect on cell growth, stable silencing of EZH2 was established in EZH2-expressing TW02 NPC cells with RNA interference. EZH2 immunolabeling was assessable for 89 primary NPC biopsy samples and correlated with clinicopathological variables, disease-specific survival (DSS), and overall survival (OS).RESULTS: Growth activity of TW02 cells was significantly suppressed (p < 0.001) with stable EZH2 silencing. Compared with normal nasopharyngeal tissue, expression levels of EZH2 transcript and protein were apparently upregulated in NPC specimens. As a continuous variable, higher EZH2 expression preferentially occurred in NPCs of T3 to T4 stages (p = 0.03) and significantly predicted inferior DSS (p = 0.0010) and OS (p = 0.004). The prognostic implications for DSS (p = 0.010) and OS (p = 0.006) still remained valid when using the median (≥60%) of EZH2 immunolabeling index to dichotomize the cohort. In the multivariate model, higher EZH2 expression was an independent adverse factor of both DSS (p = 0.012) and OS (p = 0.011), along with American Joint Committee on Cancer Stages III to IV (p = 0.024 for DSS, p = 0.017 for OS).CONCLUSION: At least partly through promoting cell growth, EZH2 implicates disease progression, confers tumor aggressiveness, and represents an independent adverse prognosticator in patients with NPC.Copyright © 2011 Elsevier Inc. All rights reserved.
|Novel control by the CA3 region of the hippocampus on neurogenesis in the dentate gyrus of the adult rat. |
Liu, JX; Pinnock, SB; Herbert, J
PloS one 6 e17562 2011
The dentate gyrus is a site of continued neurogenesis in the adult brain. The CA3 region of the hippocampus is the major projection area from the dentate gyrus. CA3 sends reciprocal projections back to the dentate gyrus. Does this imply that CA3 exerts some control over neurogenesis? We studied the effects of lesions of CA3 on neurogenesis in the dentate gyrus, and on the ability of fluoxetine to stimulate mitotic activity in the progenitor cells. Unilateral ibotenic-acid generated lesions were made in CA3. Four days later there was no change on the number of either BrdU or Ki67-positive progenitor cells in the dentate gyrus. However, after 15 or 28 days, there was a marked reduction in surviving BrdU-labelled cells on the lesioned side (but no change in Ki-67+ cells). pCREB or Wnt3a did not co-localise with Ki-67 but with NeuN, a marker of mature neurons. Lesions had no effect on the basal expression of either pCREB or Wnt3a. Subcutaneous fluoxetine (10 mg/kg/day) for 14 days increased the number of Ki67+ cells as expected on the control (non-lesioned) side but not on that with a CA3 lesion. Nevertheless, the expected increase in BDNF, pCREB and Wnt3a still occurred on the lesioned side following fluoxetine treatment. Fluoxetine has been reported to decrease the number of "mature" calbindin-positive cells in the dentate gyrus; we found this still occurred on the side of a CA3 lesion. We then showed that the expression GAP-43 was reduced in the dentate gyrus on the lesioned side, confirming the existence of a synaptic connection between CA3 and the dentate gyrus. These results show that CA3 has a hitherto unsuspected role in regulating neurogenesis in the dentate gyrus of the adult rat.Full Text Article
|Prognostic implication of MET overexpression in myxofibrosarcomas: an integrative array comparative genomic hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemical analysis. |
Lee, JC; Li, CF; Fang, FM; Wang, JW; Jeng, YM; Yu, SC; Lin, YT; Wu, JM; Tsai, JW; Li, SH; Huang, HY
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 23 1379-92 2010
It remains obscure in myxofibrosarcoma about the basis of tumorigenesis, progression, and metastasis. Chromosome 7 gains are common in some sarcomas, including myxofibrosarcoma, whereas the specific oncogenes are yet to be characterized. We performed an integrative study of MET gene at 7q31.2 to elucidate its implication in myxofibrosarcoma. Focused on candidate oncogenes on chromosome 7, 385K array comparative genomic hybridization was used to profile DNA copy number alterations of 12 samples. MET transcript was successfully quantified by real-time RT-PCR for 16 laser-microdissected tumors and two myxofibrosarcoma cell lines (NMFH-1, OH931). MET immunoexpression was assessable in 86 primary localized tumors with follow-up. To analyze endogenous MET expression and activation, NMFH-1 and OH931 cells, both with wild-type MET gene, were subjected to Western blotting and hepatocyte growth factor-treated NMFH-1 cells were evaluated for the kinetics of MET tyrosine phosphorylation. Non-random large-scale gains on 7q were detected in five cases, delineating three recurrent amplicons, 7q21.11-7q21.3, 7q22.1-22.3, and 7q31.1-7q32.3, in which the locus of MET displayed increased copy number, among others. MET mRNA was upregulated in OH931, NMFH-1, and nine tumors (56%), whereas neither gene dosage nor mRNA expression of MET was associated with clinicopathological factors. In contrast, MET protein overexpression, present in 67% of cases, was highly related to deep location (P=0.004), higher grades (P=0.001), and more advanced stages (Pless than 0.001). Importantly, MET overexpression independently portended inferior metastasis-free survival (P=0.004) and overall survival (P=0.0221). Expressing activating phospho-MET at Tyr(1234)/Tyr(1235), OH931 cells had more abundant total MET than NMFH-1 cells, whereas the latter became promptly phosphorylated on stimulation of hepatocyte growth factor. In primary myxofibrosarcomas, MET overexpression, as a frequent event, is likely driven by 7q gains with mRNA upregulation, associated with important prognosticators, and independently predictive of worse outcomes, highlighting its possible causative function in tumor aggressiveness and potentiality as a therapeutic target.
|Molecular regulation of the developing commissural plate. |
Moldrich, RX; Gobius, I; Pollak, T; Zhang, J; Ren, T; Brown, L; Mori, S; De Juan Romero, C; Britanova, O; Tarabykin, V; Richards, LJ
The Journal of comparative neurology 518 3645-61 2010
Coordinated transfer of information between the brain hemispheres is essential for function and occurs via three axonal commissures in the telencephalon: the corpus callosum (CC), hippocampal commissure (HC), and anterior commissure (AC). Commissural malformations occur in over 50 human congenital syndromes causing mild to severe cognitive impairment. Disruption of multiple commissures in some syndromes suggests that common mechanisms may underpin their development. Diffusion tensor magnetic resonance imaging revealed that forebrain commissures crossed the midline in a highly specific manner within an oblique plane of tissue, referred to as the commissural plate. This specific anatomical positioning suggests that correct patterning of the commissural plate may influence forebrain commissure formation. No analysis of the molecular specification of the commissural plate has been performed in any species; therefore, we utilized specific transcription factor markers to delineate the commissural plate and identify its various subdomains. We found that the mouse commissural plate consists of four domains and tested the hypothesis that disruption of these domains might affect commissure formation. Disruption of the dorsal domains occurred in strains with commissural defects such as Emx2 and Nfia knockout mice but commissural plate patterning was normal in other acallosal strains such as Satb2(-/-). Finally, we demonstrate an essential role for the morphogen Fgf8 in establishing the commissural plate at later developmental stages. The results demonstrate that correct patterning of the commissural plate is an important mechanism in forebrain commissure formation.
|Chondroitinase ABC enhances pericontusion axonal sprouting but does not confer robust improvements in behavioral recovery. |
Harris, NG; Mironova, YA; Hovda, DA; Sutton, RL
Journal of neurotrauma 27 1971-82 2010
Traumatic brain injury (TBI) results in enduring functional deficits. Strategies aimed at promoting plasticity within the injured brain may aid in enhancing functional outcome. We have previously shown that spontaneous pericontusional axon sprouting occurs within 7-14 days after controlled cortical impact injury in the adult rat, but ultimately fails due to an increasingly growth-inhibitory environment. We therefore sought to determine whether acute infusion of chondroitinase ABC into the site of the cortical contusion, to further reduce pericontusional growth-inhibitory chondroitin sulfate proteoglycans (CSPGs), would enhance and prolong the sprouting response. We also wanted to determine if chondroitinase-enhanced sprouting would ameliorate the behavioral deficits in forelimb function that occur in this model. Acute chondroitinase infusion decreased intact CSPGs and significantly increased pericontusional cortical grey and white matter growth-associated protein 43 (GAP43)-positive axon sprouting at 7 days post-injury. A return of intact CSPGs at later time points likely contributed to the absence of persistently increased levels of axon sprouting by 14-21 days post-injury. There was no overall benefit on forelimb function during the time of maximal sprouting or at any subsequent times in three of four behavioral outcome measures. However, there was a chondroitinase-induced improvement in recovery from unskilled limb use deficits on the staircase forelimb reaching test toward sham-injured values at 28 days, which was not achieved by the vehicle-treated rats, indicating that there is some minor functional benefit of the increased sprouting induced by chondroitinase treatment. The current results, together with data from spinal cord injury models after chondroitinase intervention, suggest that a combinatorial approach with the addition of neurotrophins and rehabilitation would result in more robust axon sprouting and consequently improve behavioral outcome.
|MMP-2 is involved in synaptic remodeling after cochlear lesion. |
Fredrich M, Illing RB
Neuroreport 21 324-7 2010
Lesion-induced neuroplasticity, including fiber degeneration, axonal growth, and synaptogenesis, involves dynamical changes of the extracellular matrix. We discovered that the matrix metalloprotease-2 (MMP-2), a major actor in extracellular matrix recomposition, is changed in distribution and increased in amount in the ventral cochlear nucleus after unilateral cochlear ablation. There was a remarkable coincidence of MMP-2 accumulation and GAP-43 expression in time and space. We obtained evidence indicating that MMP-2 is delivered to regions of emerging GAP-43 positive synaptic endings by postsynaptic neurons as well as by adjoining astrocytes. These results indicate a major role of MMP-2 in lesion-induced remodeling of central auditory networks and suggest a cooperativity with GAP-43-directed axonal outgrowth and synaptogenesis.
|Bone marrow stem cell transplantation to olfactory epithelium. |
Naoki Ochi,Kiyoshi Doi,Masahiro Uranagase,Tasuku Nishikawa,Sayaka Katsunuma,Ken-ichi Nibu
The Annals of otology, rhinology, and laryngology 119 2010
We sought to develop a new therapeutic strategy for degeneration of olfactory receptor neurons (ORNs).
|Olfactory mucosal transplantation after spinal cord injury improves voiding efficiency by suppressing detrusor-sphincter dyssynergia in rats. |
Jiro Nakayama,Tetsuya Takao,Hiroshi Kiuchi,Keisuke Yamamoto,Shinichiro Fukuhara,Yasushi Miyagawa,Masanori Aoki,Koichi Iwatsuki,Toshiki Yoshimine,Masaki Ueno,Toshihide Yamashita,Norio Nonomura,Akira Tsujimura,Akihiko Okuyama
The Journal of urology 184 2010
Several recent studies showed that olfactory mucosal transplantation after spinal cord injury promotes extensive regeneration of the injured spinal cord. We examined the efficacy of olfactory mucosal transplantation for bladder dysfunction after spinal cord injury in rats.
|Fate mapping of mammalian embryonic taste bud progenitors. |
Thirumangalathu, S; Harlow, DE; Driskell, AL; Krimm, RF; Barlow, LA
Development (Cambridge, England) 136 1519-28 2009
Mammalian taste buds have properties of both epithelial and neuronal cells, and are thus developmentally intriguing. Taste buds differentiate at birth within epithelial appendages, termed taste papillae, which arise at mid-gestation as epithelial thickenings or placodes. However, the embryonic relationship between placodes, papillae and adult taste buds has not been defined. Here, using an inducible Cre-lox fate mapping approach with the ShhcreER(T2) mouse line, we demonstrate that Shh-expressing embryonic taste placodes are taste bud progenitors, which give rise to at least two different adult taste cell types, but do not contribute to taste papillae. Strikingly, placodally descendant taste cells disappear early in adult life. As placodally derived taste cells are lost, we used Wnt1Cre mice to show that the neural crest does not supply cells to taste buds, either embryonically or postnatally, thus ruling out a mesenchymal contribution to taste buds. Finally, using Bdnf null mice, which lose neurons that innervate taste buds, we demonstrate that Shh-expressing taste bud progenitors are specified and produce differentiated taste cells normally, in the absence of gustatory nerve contact. This resolution of a direct relationship between embryonic taste placodes with adult taste buds, which is independent of mesenchymal contribution and nerve contact, allows us to better define the early development of this important sensory system. These studies further suggest that mammalian taste bud development is very distinct from that of other epithelial appendages.
|IFATS collection: Human adipose tissue-derived stem cells induce angiogenesis and nerve sprouting following myocardial infarction, in conjunction with potent preservation of cardiac function. |
Cai, L; Johnstone, BH; Cook, TG; Tan, J; Fishbein, MC; Chen, PS; March, KL
Stem cells (Dayton, Ohio) 27 230-7 2009
The administration of therapeutic cell types, such as stem and progenitor cells, has gained much interest for the limitation or repair of tissue damage caused by a variety of insults. However, it is still uncertain whether the morphological and functional benefits are mediated predominantly via cell differentiation or paracrine mechanisms. Here, we assessed the extent and mechanisms of adipose-derived stromal/stem cells (ASC)-dependent tissue repair in the context of acute myocardial infarction. Human ASCs in saline or saline alone was injected into the peri-infarct region in athymic rats following left anterior descending (LAD) coronary artery ligation. Cardiac function and structure were evaluated by serial echocardiography and histology. ASC-treated rats consistently exhibited better cardiac function, by all measures, than control rats 1 month following LAD occlusion. Left ventricular (LV) ejection fraction and fractional shortening were improved in the ASC group, whereas LV remodeling and dilation were limited in the ASC group compared with the saline control group. Anterior wall thinning was also attenuated by ASC treatment, and post-mortem histological analysis demonstrated reduced fibrosis in ASC-treated hearts, as well as increased peri-infarct density of both arterioles and nerve sprouts. Human ASCs were persistent at 1 month in the peri-infarct region, but they were not observed to exhibit significant cardiomyocyte differentiation. Human ASCs preserve heart function and augment local angiogenesis and cardiac nerve sprouting following myocardial infarction predominantly by the provision of beneficial trophic factors.
|Expression and localization of the calmodulin-binding protein neurogranin in the adult mouse olfactory bulb. |
S Gribaudo,S Bovetti,D Garzotto,A Fasolo,S De Marchis
The Journal of comparative neurology 517 2009
Neurogranin (Ng) is a brain-specific postsynaptic protein involved in activity-dependent synaptic plasticity through modulation of Ca(2+)/calmodulin (CaM)-dependent signal transduction in neurons. In this study, using biochemical and immunohistochemical approaches, we demonstrate Ng expression in the adult mouse olfactory bulb (OB), the first relay station in odor information processing. We show that Ng is principally associated with the granule cell layer (GCL), which is composed of granule cell inhibitory interneurons. This cell type is continuously renewed during adult life and plays a key role in OB circuits, integrating and modulating the activity of mitral/tufted cells. Our results indicate that Ng localizes in the soma and dendrites of a defined subpopulation of mature GABAergic granule cells, enriched in the deep portion of the GCL. Ng-immunopositive cells largely coexpress the Ca(+)/CaM-dependent kinase IV (CaMKIV), a downstream protein of CaM signaling cascade, whereas no colocalization was observed between Ng and the calcium-binding protein calretinin. Finally, we demonstrate that adult neurogenesis contributes to the Ng-expressing population, with more newly generated Ng-positive cells integrated in the deep GCL. Together, these results provide a new specific neurochemical marker to identify a subpopulation of olfactory granule cells and suggest possible functional implications for Ng in OB plasticity mechanisms.
|Phosphodiesterase 1C is dispensable for rapid response termination of olfactory sensory neurons. |
Cygnar, KD; Zhao, H
Nature neuroscience 12 454-62 2009
In the nose, odorants are detected on the cilia of olfactory sensory neurons (OSNs), where a cAMP-mediated signaling pathway transforms odor stimulation into electrical responses. Phosphodiesterase (PDE) activity in OSN cilia has long been thought to account for rapid response termination by degrading odor-induced cAMP. Two PDEs with distinct cellular localization have been found in OSNs: PDE1C in the cilia and PDE4A throughout the cell but absent from the cilia. We disrupted both of these genes in mice and carried out electro-olfactogram analysis. Unexpectedly, eliminating PDE1C did not prolong response termination. Prolonged termination occurred only in mice that lacked both PDEs, suggesting that cAMP degradation by PDE1C in cilia is not a rate-limiting factor for response termination in wild-type mice. Pde1c(-/-) OSNs instead showed reduced sensitivity and attenuated adaptation to repeated stimulation, suggesting that PDE1C may be involved in regulating sensitivity and adaptation. Our observations provide new perspectives on the regulation of olfactory transduction.Full Text Article
|Effect of intranasal administration of basic fibroblast growth factor on olfactory epithelium. |
Tasuku Nishikawa, Kiyoshi Doi, Naoki Ochi, Sayaka Katsunuma, Ken-ichi Nibu, Tasuku Nishikawa, Kiyoshi Doi, Naoki Ochi, Sayaka Katsunuma, Ken-Ichi Nibu, Tasuku Nishikawa, Kiyoshi Doi, Naoki Ochi, Sayaka Katsunuma, Ken-ichi Nibu
Neuroreport 20 764-9 2009
To further study the effects of basic fibroblast growth factor (bFGF) on the olfactory epithelium, bFGF was intranasally administered twice a day for 6 weeks to 2.5-month-old and 7-month-old mice. The effects were immunohistochemically examined by using antibodies against proliferating cell nuclear antigen, olfactory marker protein, and GAP43. The number of cells positive for proliferating cell nuclear antigen in the supporting cell layer increased dramatically, and that of GAP43-positive cells, or globose basal cells, increased significantly, especially in aging mice. However, no significant changes were observed in the number of olfactory marker protein-positive cells or mature olfactory receptor neurons. These results suggest that topical application of bFGF promotes proliferation of globose basal cells and supporting cells.
|Central auditory plasticity after carboplatin-induced unilateral inner ear damage in the chinchilla: Up-regulation of GAP-43 in the ventral cochlear nucleus. |
K S Kraus, D Ding, Y Zhou, R J Salvi, K S Kraus, D Ding, Y Zhou, R J Salvi
Hearing research 255 33-43 2009
Inner ear damage may lead to structural changes in the central auditory system. In rat and chinchilla, cochlear ablation and noise trauma result in fiber growth and synaptogenesis in the ventral cochlear nucleus (VCN). In this study, we documented the relationship between carboplatin-induced hair cell degeneration and VCN plasticity in the chinchilla. Unilateral application of carboplatin (5mg/ml) on the round window membrane resulted in massive hair cell loss. Outer hair cell degeneration showed a pronounced basal-to-apical gradient while inner hair cell loss was more equally distributed throughout the cochlea. Expression of the growth associated protein GAP-43, a well-established marker for synaptic plasticity, was up-regulated in the ipsilateral VCN at 15 and 31 days post-carboplatin, but not at 3 and 7 days. In contrast, the dorsal cochlear nucleus showed only little change. In VCN, the high-frequency area dorsally showed slightly yet significantly stronger GAP-43 up-regulation than the low-frequency area ventrally, possibly reflecting the high-to-low frequency gradient of hair cell degeneration. Synaptic modification or formation of new synapses may be a homeostatic process to re-adjust mismatched inputs from two ears. Alternatively, massive fiber growth may represent a deleterious process causing central hyperactivity that leads to loudness recruitment or tinnitus.
|Lack of axonal sprouting of spared propriospinal fibers caudal to spinal contusion injury is attributed to chronic axonopathy. |
AC Steencken, JR Siebert, DJ Stelzner
Journal of neurotrauma 26 2279-97 2009
We have previously shown that a small percentage of long descending propriospinal tract (LDPT) axons are spared, whereas few short thoracic propriospinal (TPS) fibers survive 2 weeks following severe (50 mm weight drop) low thoracic spinal cord contusion injury (SCI). Here, we extended those findings to a moderate (25 mm weight drop) T9 SCI and assessed the effects of this lesion severity on propriospinal tract fibers at different time periods after injury. We anterogradely labeled fibers with fluororuby (FR) or WGA-HRP to determine their location and number 2, 4, 6, and 16 weeks post-SCI. Findings were compared with non-injured controls. At chronic time points, surviving FR-labeled LDPT fibers rostral to the injury remained as reactive endings or as putative regenerative sprouts. Caudal to the injury, spared LDPT fibers ran along a rim of lateral and ventral white matter, and ended as small abnormal-appearing putative terminal boutons or reactive endings within the intermediate gray matter of lumbosacral cord, with little axonal arborization and no evidence of injury-induced sprouting. One striking difference in the WGA-HRP experimental operates was the increased density of labeling of spared axons within the white matter caudal to the injury compared to controls. This labeling pattern was reminiscent of the labeling found after axotomy in studies by others, and raises a question as to contusion injury-induced impaired axonal transport. We hypothesize that axonal sprouting of axons after partial spinal cord injury seen in previous investigations was not found in the present investigation because of the additional pathological effects of contusion injury, similar to what is observed after traumatic brain injury.
|Ezrin overexpression in gastrointestinal stromal tumors: an independent adverse prognosticator associated with the non-gastric location. |
YC Wei, CF Li, SC Yu, FF Chou, FM Fang, HL Eng, YH Uen, YF Tian, JM Wu, SH Li, WW Huang, WM Li, HY Huang
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 22 1351-60 2009
Ezrin, a member of the ezrin-radixin-moesin family, acts as a link between the cell membrane and actin cytoskeleton to integrate cell adhesion-mediated signaling. It implicates tumor progression, metastatic dissemination, and adverse outcomes in several cancer types, including pediatric and adult sarcomas. Although ezrin upregulation was shown by cDNA expression profiling, no study has systematically evaluated the significance of ezrin expression in a large cohort of gastrointestinal stromal tumors (GISTs). Ezrin immunostaining was carried out on tissue microarrays of primary GISTs and assessable in 347 cases, 188 of which were successfully evaluated for mutation variants of KIT and PDGFRA receptor tyrosine kinase (RTK) genes by sequencing with or without screening by denatured high-performance liquid chromatography. These GISTs with known RTK genotypes were dichotomized into two prognostically different groups. The endogenous expression and phosphorylation of ezrin in GIST cell lines were analyzed by western blotting. By immunohistochemistry, ezrin overexpression was present in 66% of GISTs and significantly associated with the non-gastric location (P=0.002) and decreased disease-free survival (P=0.032, univariately). However, it was not related to the National Institute of Health (NIH) risk category, Ki-67 labeling index, RTK genotypes, and other variables. In multivariate analyses, ezrin overexpression remained independently predictive of adverse outcome (P=0.008, risk ratio=2.363), together with Ki-67 labeling index >5% (P<0.001, risk ratio=3.581), high-risk category (P<0.001, risk ratio=2.156), and the non-gastric location (P=0.029, risk ratio=1.899). Despite the variation in the ezrin expression level, phosphorylated ezrin at threonine(567) was only detectable in GIST882 and GIST48 cells, but not in colonic smooth muscle cells. In conclusion, ezrin is frequently overexpressed in GISTs, especially those arising from the non-gastric sites. Given that its impact is independent of the NIH risk category, cell proliferation, and tumor location, ezrin immunoreactivity represents a valuable prognostic adjunct of GISTs, suggesting a causative role in conferring an aggressive phenotype.
|Heat shock protein 90 overexpression independently predicts inferior disease-free survival with differential expression of the alpha and beta isoforms in gastrointestinal stromal tumors. |
Li, CF; Huang, WW; Wu, JM; Yu, SC; Hu, TH; Uen, YH; Tian, YF; Lin, CN; Lu, D; Fang, FM; Huang, HY
Clinical cancer research : an official journal of the American Association for Cancer Research 14 7822-31 2008
Most gastrointestinal stromal tumors harbor a mutated KIT or PDGFRA receptor tyrosine kinase (RTK). Heat shock protein 90 (Hsp90) is a chaperone mediating the folding and stabilization of many oncoproteins, including KIT. An Hsp90 inhibitor, 17-AAG, can attenuate KIT activation and proliferation of gastrointestinal stromal tumor cell lines. We further evaluated Hsp90 immunoexpression and the difference between alpha and beta isoforms in gastrointestinal stromal tumor specimens.Hsp90 immunostain was assessable in 306 cases on tissue microarrays of primary gastrointestinal stromal tumors and correlated with various variables and disease-free survival (DFS). RTK mutation variants, confirmed in 142 cases by sequencing with or without precedent denaturing high pressure liquid chromatography screening, were dichotomized into two prognostically different groups. Differential expression of transcript and protein isoforms was measured by real-time reverse transcription-PCR and Western blotting in 16 and 6 cases, respectively.Hsp90 overexpression (55%) significantly correlated with larger size, nongastric location, higher mitotic count and NIH risk level, Ki-67 overexpression (all P less than or = 0.001), and unfavorable RTK genotypes (P = 0.020). It strongly portended inferior DFS univariately (P less than 0.0001) and remained independent in multivariate analysis (P = 0.031; risk ratio, 2.44), along with high-risk category, Ki-67 overexpression, and old age. For both mRNA and protein, Hsp90beta was more abundant than Hsp90alpha, whereas the latter was significantly higher in high-risk cases.Hsp90 overexpression represents a poor prognosticator that correlates with several adverse parameters, highlighting its role in disease progression and alternative therapy for high-risk, imatinib-resistant gastrointestinal stromal tumors. Hsp90alpha seems more relevant to the intrinsic aggressiveness of gastrointestinal stromal tumors, albeit less abundant than Hsp90beta.
|Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia. |
Tom Chao,Khoa Pham,Oswald Steward,Ranjan Gupta
The Journal of comparative neurology 506 2008
Chronic nerve compression (CNC) injury initiates a series of pathological changes within the peripheral nerve at the site of injury. However, to date, little work has been performed to explore neuronal cell body responses to CNC injury. Here we show a preferential upregulation of growth-associated protein-43 (GAP-43) and enhanced Fluoro Ruby uptake by the small-diameter calcitonin gene-related protein (CGRP) and isolectin B4 (IB4)-positive neurons in the L4 and L5 ipsilateral dorsal root ganglion (DRG) 2 weeks and 1 month post injury. Furthermore, L4 and L5 DRGs ipsilateral to CNC injury also demonstrated a marked reduction in neurofilament 200 (NF-200) neurons and an increase in CGRP and IB4 neurons at early time points. All numbers normalized to values comparable to those of control when the DRG was evaluated 6 months post injury. Quantification of glial-derived neurotrophic factor (GDNF) protein revealed an upregulation in L4 and L5 DRG followed by a return to baseline values at later stages following injury. Upregulation of GDNF expression by Schwann cells was also readily apparent with both immunohistochemistry and Western blot analysis of 1 month compressed sciatic nerve specimens. Thus, CNC induces a phenotypic change in the DRG that appears to be temporally associated with increases in GDNF protein expression at and near the site of the compression injury in the nerve.
|Cardiac sympathetic rejuvenation: a link between nerve function and cardiac hypertrophy. |
Kimura, K; Ieda, M; Kanazawa, H; Yagi, T; Tsunoda, M; Ninomiya, S; Kurosawa, H; Yoshimi, K; Mochizuki, H; Yamazaki, K; Ogawa, S; Fukuda, K
Circulation research 100 1755-64 2007
Neuronal function and innervation density is regulated by target organ-derived neurotrophic factors. Although cardiac hypertrophy drastically alternates the expression of various growth factors such as endothelin-1, angiotensin II, and leukemia inhibitory factor, little is known about nerve growth factor expression and its effect on the cardiac sympathetic nerves. This study investigated the impact of pressure overload-induced cardiac hypertrophy on the innervation density and cellular function of cardiac sympathetic nerves, including kinetics of norepinephrine synthesis and reuptake, and neuronal gene expression. Right ventricular hypertrophy was induced by monocrotaline treatment in Wistar rats. Newly developed cardiac sympathetic nerves expressing beta(3)-tubulin (axonal marker), GAP43 (growth-associated cone marker), and tyrosine hydroxylase were markedly increased only in the right ventricle, in parallel with nerve growth factor upregulation. However, norepinephrine and dopamine content was paradoxically attenuated, and the protein and kinase activity of tyrosine hydroxylase were markedly downregulated in the right ventricle. The reuptake of [(125)I]-metaiodobenzylguanidine and [(3)H]-norepinephrine were also significantly diminished in the right ventricle, indicating functional downregulation in cardiac sympathetic nerves. Interestingly, we found cardiac sympathetic nerves in hypertrophic right ventricles strongly expressed highly polysialylated neural cell adhesion molecule (PSA-NCAM) (an immature neuron marker) as well as neonatal heart. Taken together, pressure overload induced anatomical sympathetic hyperinnervation but simultaneously caused deterioration of neuronal cellular function. This phenomenon was explained by the rejuvenation of cardiac sympathetic nerves as well as the hypertrophic cardiomyocytes, which also showed the fetal form gene expression.
|A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. |
Meikle, L; Talos, DM; Onda, H; Pollizzi, K; Rotenberg, A; Sahin, M; Jensen, FE; Kwiatkowski, DJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 5546-58 2007
Tuberous sclerosis (TSC) is a hamartoma syndrome caused by mutations in TSC1 or TSC2 in which cerebral cortical tubers and seizures are major clinical issues. We have engineered mice in which most cortical neurons lose Tsc1 expression during embryonic development. These Tsc1 mutant mice display several neurological abnormalities beginning at postnatal day 5 with subsequent failure to thrive and median survival of 35 d. The mice also display clinical and electrographic seizures both spontaneously and with physical stimulation, and some seizures end in a fatal tonic phase. Many cortical and hippocampal neurons are enlarged and/or dysplastic in the Tsc1 mutant mice, strongly express phospho-S6, and are ectopic in multiple sites in the cortex and hippocampus. There is a striking delay in myelination in the mutant mice, which appears to be caused by an inductive neuronal defect. This new TSC brain model replicates several features of human TSC brain lesions and implicates an important function of Tsc1/Tsc2 in neuronal development.
|Late effects of enriched environment (EE) plus multimodal early onset stimulation (MEOS) after traumatic brain injury in rats: Ongoing improvement of neuromotor function despite sustained volume of the CNS lesion. |
Marcela Lippert-Gruener, Marc Maegele, Janika Garbe, Doychin N Angelov
Experimental neurology 203 82-94 2007
Recently we showed that the combination between MEOS and EE applied to rats for 7-15 days after traumatic brain injury (TBI) was associated with reduced CNS lesion volume and enhanced reversal of neuromotor dysfunction. In a continuation of this work, we tested whether these effects persisted for longer post-operative periods, e.g. 30 days post-injury (dpi). Rats were subjected to lateral fluid percussion (LFP) or to sham injury. After LFP, one third of the animals (injured and sham) was placed under conditions of standard housing (SH), one third was kept in EE-only, and one third received EE+MEOS. Standardized composite neuroscore (NS) for neurological functions and computerized analysis of the vibrissal motor performance were used to assess post-traumatic neuromotor deficits. These were followed by evaluation of the cortical lesion volume (CLV) after immunostaining for neuron-specific enolase, caspase 3 active, and GFAP. Finally, the volume of cortical lesion containing regeneration-associated proteins (CLV-RAP) was determined in sections stained for GAP-43, MAP2, and neuronal class III beta-tubulin. We found (i) no differences in the vibrissal motor performance; (ii) EE+MEOS rats performed significantly better than SH rats in NS; (iii) EE-only and EE+MEOS animals, but not SH rats, showed better recovery at 30 dpi than at 15 dpi; (iv) no differences among all groups in CLV (larger than that at 15 dpi) and CLV-RAP, despite a clear tendency to reduction in the EE-only and EE+MEOS rats. We conclude that EE+MEOS retards, but cannot prevent the increase of lesion volume. This retardation is sufficient for a continuous restoration of neurological functions.
|Hardwiring the brain: endocannabinoids shape neuronal connectivity. |
Berghuis, Paul, et al.
Science, 316: 1212-6 (2007) 2007
The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB(1) cannabinoid receptors (CB(1)Rs) are enriched in the axonal growth cones of gamma-aminobutyric acid-containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB(1)R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB(1)Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.
|Evaluation of olfactory ensheathing and schwann cells after implantation into a dorsal injury of adult rat spinal cord. |
Melissa R Andrews, Dennis J Stelzner
Journal of neurotrauma 24 1773-92 2007
Olfactory ensheathing cells (OECs) and Schwann cells (SCs) obtained from adult transgenic rats expressing alkaline phosphatase (AP) were studied following implantation into intact spinal cord and after dorsal column crush (DCC) injury, either within the lesion or near the lesion borders. We observed no evidence of migration of AP OECs or AP SCs after lesion site injections, with most cells remaining in or nearby the injection/lesion site. Acute injection of either cell type outside of the lesion site resulted in the presence of cells in the lesion even two hours after injection. However, after a 2-week delay between DCC injury and cell injection, only OECs injected 2.5-mm outside of a DCC lesion entered the lesion, while SCs did not pass a region of increased astroglial immunoreactivity. GFAP-immunoreactivity also revealed differences in the astroglial scar at the lesion border with openings apparent in this region only in the OEC group. SCs induced greater ingrowth of CGRP-positive axons within the lesion, two weeks post-injury. Equivalent numbers of GAP-43-positive axons grew within the lesion after SC or OEC implantation. These findings show that, although there is no active migration for either cell type, both OECs and SCs are able to support axonal regrowth and/or sprouting into the lesion. The openings in the astroglial boundary at the lesion site may give OECs a potential advantage over SCs in promoting axonal growth through the astroglial scar.
|Musashi-1 expression in postnatal mouse olfactory epithelium. |
Kenta Watanabe, Kenji Kondo, Naonobu Takeuchi, Hideyuki Okano, Tatsuya Yamasoba, Kenta Watanabe, Kenji Kondo, Naonobu Takeuchi, Hideyuki Okano, Tatsuya Yamasoba
Neuroreport 18 641-4 2007
We investigated the age-related change in the distribution of a molecular marker for neural stem and precursor cells, Musashi-1, in the olfactory epithelium of mice from 1 day up to 16 months of age using immunohistochemistry. We also compared the distribution pattern of Musashi-1 with that of growth-associated protein 43, the olfactory marker protein, and Notch-1. Musashi-1 was expressed in the globose basal cell layer and the lower part of the growth-associated protein 43-positive layer, with immunoreactivity decreasing with aging. Notch-1 was observed only in the early postnatal period. These findings are consistent with the fact that globose basal cells are proliferating olfactory precursor cells and that their ability to generate new neurons decreases with aging.
|Calsyntenin-1 docks vesicular cargo to kinesin-1. |
Konecna, A; Frischknecht, R; Kinter, J; Ludwig, A; Steuble, M; Meskenaite, V; Indermühle, M; Engel, M; Cen, C; Mateos, JM; Streit, P; Sonderegger, P
Molecular biology of the cell 17 3651-63 2006
We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1-containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.
|Cochlear damage induces GAP-43 expression in cholinergic synapses of the cochlear nucleus in the adult rat: a light and electron microscopic study |
Meidinger, M. A., et al.
Eur. J. Neurosci., 23:3187-3199 (2006) 2006
|Growth-associated gene and protein expression in the region of axonal sprouting in the aged brain after stroke. |
Songlin Li, S Thomas Carmichael
Neurobiology of disease 23 362-73 2006
Aged individuals exhibit reduced functional recovery after stroke. We examined the expression profile in aged animals of a recently identified group of growth-associated genes that underlies post-stroke axonal sprouting in the young adult. Basal levels of most growth-promoting genes are higher in aged cortex compared with young adult, and are further induced after stroke. Compared with the young adult, these genes are induced at later time points after stroke. For growth-inhibitory molecules, myelin-associated glycoprotein and ephrin A5 are uniquely induced in the aged brain; chondroitin sulfate proteoglycans and oligodendrocyte myelin glycoprotein are induced at earlier time points; and Nogo-A, semaphorin IIIa and NG2 decline in aged vs. young adult after stroke. The aged brain does not simply have a reduction in growth-associated molecules after stroke, but a completely unique molecular profile of post-stroke axonal sprouting.
|Skp2 overexpression is highly representative of intrinsic biological aggressiveness and independently associated with poor prognosis in primary localized myxofibrosarcomas. |
Huang, HY; Kang, HY; Li, CF; Eng, HL; Chou, SC; Lin, CN; Hsiung, CY
Clinical cancer research : an official journal of the American Association for Cancer Research 12 487-98 2006
Two SCF(Skp2) ubiquitin ligase-related proteins, Skp2 and cyclin-dependent kinase subunit 1 (Cks1), are involved in posttranscriptional degradation of p27(Kip1) tumor suppressor. We analyzed the prognostic utility of p27(Kip1) and its interacting cell cycle regulators in myxofibrosarcomas.Clinicopathologic features and tissue microarray-based immunohistochemical expression of p27(Kip1), Skp2, Cks1, cyclin E, cyclin A, Ki-67, and minichromosome maintenance protein 2 (Mcm2) were assessed in 70 primary myxofibrosarcomas and correlated with clinical outcomes. Skp2 mRNA expression and the relationship between Skp2 and p27(Kip1) proteins were examined in six cases by semiquantitative reverse transcription-PCR and Western blotting, respectively.High indices of Skp2 (greater than or =10%), cyclin A (greater than or =10%), and Mcm2 (greater than or =50%) were adverse prognosticators at the univariate level. Furthermore, co-overexpression of Skp2 and cyclin A identified highly lethal cases in the entire cohort [P less than 0.0001 for disease-specific survival (DSS), P = 0.0004 for overall survival (OS)] and the lower-grade subset (Fédération Nationale des Centres de Lutte Contre le Cancer grade 1 and 2; P = 0.0006 for DSS, P = 0.0093 for OS). In multivariate analyses, Skp2 overexpression overshadowed most intrinsic clinicopathologic factors and independently correlated with worse metastasis-free survival (P = 0.0012), DSS (P = 0.0234), and OS (P = 0.0056). Notably, positive margins independently predicted inferior local recurrence-free survival (P = 0.0012) and also negatively affected metastasis-free survival (P = 0.0471), DSS (P = 0.0152), and OS (P = 0.0173). Reverse transcription-PCR showed up-regulation of Skp2 mRNA in four cases and Western blotting displayed a matched expression pattern of Skp2.Margin status and intrinsic property of myxofibrosarcomas both affect patient survival. Skp2 overexpression is highly representative of the biological aggressiveness of myxofibrosarcomas and plays an important prognostic role.
|Chondroitinase ABC combined with neural stem/progenitor cell transplantation enhances graft cell migration and outgrowth of growth-associated protein-43-positive fibers after rat spinal cord injury. |
Takeshi Ikegami, Masaya Nakamura, Junichi Yamane, Hiroyuki Katoh, Seiji Okada, Akio Iwanami, Kota Watanabe, Ken Ishii, Fumikazu Kato, Hiroshi Fujita, Toyomi Takahashi, Hirotaka James Okano, Yoshiaki Toyama, Hideyuki Okano
The European journal of neuroscience 22 3036-46 2005
We previously reported that the transplantation of neural stem/progenitor cells (NSPCs) can contribute to the repair of injured spinal cord in adult rats and monkeys. In some cases, however, most of the transplanted cells adhered to the cavity wall and failed to migrate and integrate into the host spinal cord. In this study we focused on chondroitin sulfate proteoglycan (CSPG), a known constituent of glial scars that is strongly expressed after spinal cord injury (SCI), as a putative inhibitor of NSPC migration in vivo. We hypothesized that the digestion of CSPG by chondroitinase ABC (C-ABC) might promote the migration of transplanted cells and neurite outgrowth after SCI. An in vitro study revealed that the migration of NSPC-derived cells was inhibited by CSPG and that this inhibitory effect was attenuated by C-ABC pre-treatment. Consistently, an in vivo study of C-ABC treatment combined with NSPC transplantation into injured spinal cord revealed that C-ABC pre-treatment promoted the migration of the transplanted cells, whereas CSPG-immunopositive scar tissue around the lesion cavity prevented their migration into the host spinal cord in the absence of C-ABC pre-treatment. Furthermore, this combined treatment significantly induced the outgrowth of a greater number of growth-associated protein-43-positive fibers at the lesion epicentre, compared with NSPC transplantation alone. These findings suggested that the application of C-ABC enhanced the benefits of NSPC transplantation for SCI by reducing the inhibitory effects of the glial scar, indicating that this combined treatment may be a promising strategy for the regeneration of injured spinal cord.
|Early profiles of axonal growth and astroglial response after spinal cord hemisection and implantation of Schwann cell-seeded guidance channels in adult rats. |
Jung-Yu C Hsu, Xiao-Ming Xu
Journal of neuroscience research 82 472-83 2005
We previously demonstrated that transplantation of Schwann cell-seeded channels promoted the regrowth of injured axons in the adult spinal cord. It is not clear, however, whether injured axons recapitulate the developmental scenarios to accomplish regeneration. In the present study, we investigated the early events associated with axonal regrowth after spinal cord hemisection at the eighth thoracic level and implantation of a Schwann cell-seeded minichannel in adult rats. Animals were sacrificed at postoperative days (PO) 2, 4, 7, and 14. Anterograde tracing with fluoro-ruby showed that regenerating axons grew into the graft prior to PO2 and reached the distal end of the channel at PO7. These axons expressed both embryonic neural cell adhesion molecule (E-NCAM) and growth associated protein-43 (GAP-43). Although the expression of E-NCAM decreased by PO7, that of GAP-43 remained high throughout the first 2 weeks after implantation. A close relation of vimentin-positive astroglia to the growing axons in the host tissue suggested a contact-mediated role of these cells in axon guidance. Aggregation of glial fibrillary acidic protein (GFAP)-positive astrocytes together with the increased expression of chondroitin sulfate proteoglycans (CSPGs) starting at PO7 appeared to inhibit axonal growth at the host-graft interface. Thus, adult regenerating axons and astroglia do express developmentally related molecules that may facilitate axonal growth into a permissive graft at the early phase of injury and regeneration. These results suggest that molecules and astroglia essential to development are both important in influencing axonal regrowth in the adult spinal cord.
|Coexistence of reactive plasticity and neurodegeneration in Alzheimer diseased brains. |
J Guevara, H Dilhuydy, B Espinosa, A Delacourte, R Quirion, R Mena, Y Joanette, E Zenteno, Y Robitaille
Histology and histopathology 19 1075-84 2004
Alzheimer's disease (AD) is a pathological process characterized by neuron degeneration and, as recently suggested, brain plasticity. In this work, we compared the reactive plasticity in AD brains associated to O-glycosydically linked glycans, recognized by lectins from Amaranthus leucocarpus (ALL) and Macrobrachium rosenbergii (MRL), and the tau neuritic degeneration. The neuritic degenerative process was evaluated by the quantification of aggregated neuritic structures. Lesions were determined using antibodies against hyperphosphorylated-tau (AD2), amyloid-beta, and synaptophysin. In these conditions, we classified and quantified three pathological structures associated to the neuritic degenerative process: 1) Amyloid-beta deposits (AbetaDs), 2) Classic neuritic plaques (NPs), and 3) Dystrophic neurites clusters (DNCs) lacking amyloid-beta deposits. Reactive plasticity structures were constituted by meganeuritic clusters (MCs) and peri-neuronal sprouting in neurons of the CA4 region of the hippocampus, immunoreactive to synaptophysin (exclusively in AD brains) and GAP-43. Besides, MCs were associated to sialylated O-glycosydically linked glycans as determined by positive labeling with ALL and MRL. Considering that these lectins are specific for the synaptic sprouting process in AD, our results suggest the co-occurrence of of several areas of reactive plasticity and neuron degeneration in AD.
|Notch expression in developing olfactory neuroepithelium. |
Kiyoshi Doi, Haruhiko Ishida, Ken-ichi Nibu
Neuroreport 15 945-7 2004
Notch genes encode receptors for signaling pathways that regulate neurogenesis in various tissues. To better understand the roles of Notch genes in olfactory neurogenesis, we studied the expression of Notch family in developing mouse olfactory epithelium. In the earliest stage of olfactory development, Notch1 was observed in the mesenchyme lateral to the olfactory placode. During the developmental stage, Notch1 was mainly observed around the basal membrane, while Notch3 was observed in the lower compartment of the olfactory neuroepithelium. Notch2 was not detected during the entire observation period. As the olfactory neuroepithelium grew mature, both Notch1 and Notch3 gradually disappeared. These results suggest distinct roles of Notch1 and Notch3 in the neurogenesis of the peripheral olfactory system.
|Neurofilament 200 as an indicator of differences between mipafox and paraoxon sensitivity in Sy5Y neuroblastoma cells. |
Taehyeon Cho, Evelyn Tiffany-Castiglioni
Journal of toxicology and environmental health. Part A 67 987-1000 2004
Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM mipafox during the last 5 days of neurite extension (experimental set A). However, mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs.
|Myelination triggers local loss of axonal CNR/protocadherin alpha family protein expression. |
Hirofumi Morishita, Masahumi Kawaguchi, Yoji Murata, Chika Seiwa, Shun Hamada, Hiroaki Asou, Takeshi Yagi
The European journal of neuroscience 20 2843-7 2004
The cadherin-related neuronal receptor (CNR)/protocadherin (Pcdh) alpha family is one of the diverse protocadherin families expressed in developing axons. We observed a strong axonal expression of these proteins at late embryonic and early postnatal stages corresponding to regions where fibers had not yet been myelinated. We therefore followed the postnatal localization of CNR/Pcdh alpha protein in major axonal tracts, such as the internal capsule, lateral olfactory tract, and optic nerve, and found that its axonal localization was dramatically lost in parallel with the increased expression of myelin markers. Moreover, the hypomyelinated optic nerve tracts of the myelin-deficient Shiverer mouse exhibited elevated levels of CNR/Pcdh alpha expression. These axonal expression patterns of CNR/Pcdh alpha in wild-type and Shiverer mice were similar to those of growth associated protein 43 (GAP-43) and L1, both of which are associated with axonal maturation. Thus, myelination may be a trigger for the local loss of axonal CNR/Pcdh alpha protein, and this process may be important in the maturation of neural circuits.
|Expression of axotomy-inducible and apoptosis-related genes in sensory nerves of rats with experimental diabetes. |
R C Burnand, S A Price, M McElhaney, D Barker, D R Tomlinson
Brain research. Molecular brain research 132 235-40 2004
In diabetes, peripheral nerves suffer deficient neurotrophic support-a situation which resembles axotomy. This raises the question: does inappropriate establishment of an axotomised neuronal phenotype contribute to diabetic neuropathy, and in extremis, does this provoke apoptosis? We hybridized reverse-transcribed RNA, from the dorsal root ganglia (DRG) of 8-week streptozotocin (STZ)-induced diabetic rats, to Affymetrix Rat Genome U34A chips and scanned the array for expression of (a) genes that are upregulated by axotomy, (b) proapoptotic and (c) anti-apoptotic genes. Expression of the axotomy-responsive genes coding for growth-associated protein 43 (GAP-43), galanin, neuropeptide Y (NPY), pre-pro-vasoactive intestinal polypeptide (pre-pro-VIP), neuronal nitric oxide synthase (nNOS), protease nexin 1, heat-shock protein 27 (HSP 27) and myosin light chain kinase II (MLCK II) was unaffected in ganglia from diabetic rats compared to controls; thus, no axotomised phenotype was established. The expression of the majority of proapoptotic genes in the DRG was also unaltered (bax, bad, bid, bok, c-Jun, p38, TNFR1, caspase 3 and NOS2). Similarly there was no change in expression of the majority of antiapoptotic genes (bcl2, bcl-xL, bcl-w, NfkappaB). These alterations in gene expression make it clear that neither axotomy nor apoptotic phenotypes are established in neurones in this model of diabetes.
|Presynaptic proteins in the prefrontal cortex of patients with schizophrenia and rats with abnormal prefrontal development. |
Halim, ND; Weickert, CS; McClintock, BW; Hyde, TM; Weinberger, DR; Kleinman, JE; Lipska, BK
Molecular psychiatry 8 797-810 2003
Dysfunction of the prefrontal cortex in schizophrenia may be associated with abnormalities in synaptic structure and/or function and reflected in altered concentrations of proteins in presynaptic terminals and involved in synaptic plasticity (synaptobrevin/ vesicle-associated membrane protein (VAMP), synaptosomal-associated protein-25 (SNAP-25), syntaxin, synaptophysin and growth-associated protein-43 (GAP-43)). We examined the immunoreactivity of these synapse-associated proteins via quantitative immunoblotting in the prefrontal cortex of patients with schizophrenia (n=18) and in normal controls (n=23). We also tested the stability of these proteins across successive post-mortem intervals in rat brains (at 0, 3, 12, 24, 48, and 70 h). To investigate whether experimental manipulation of prefrontal cortical development in the rat alters prefrontal synaptic protein levels, we lesioned the ventral hippocampus of rats on postnatal day 7 and measured immunoreactivity of presynaptic proteins in the prefrontal cortex on postnatal day 70. VAMP immunoreactivity was lower in the schizophrenic patients by 22% (Pless than 0.03). There were no differences in the immunoreactivity of any other proteins measured in schizophrenic patients as compared to the matched controls. Proteins were fairly stable up to 24 h and thereafter the abundance of most proteins examined was significantly reduced (falling to as low as 20% of baseline levels at 48-70 h). VAMP immunoreactivity was higher in the lesioned rats as compared to sham controls by 22% (P&less than 0.03). There were no significant differences between the lesioned rats and sham animals in any other presynaptic protein. These data suggest that apparently profound prefrontal cortical dysfunction in schizophrenia, as well as in an animal model of schizophrenia, may exist without gross changes in the abundance of many synaptic proteins but discrete changes in selected presynaptic molecules may be present.
|Spatiotemporal distribution of neuronal calcium sensor-1 in the developing rat spinal cord. |
Takayuki Kawasaki, Takeshi Nishio, Hisashi Kurosawa, John Roder, Andreas Jeromin
The Journal of comparative neurology 460 465-75 2003
The present study revealed the localization of neuronal calcium sensor (NCS)-1 immunoreactivity (IR) in the developing rat spinal cord. The NCS-1 IR first appeared at embryonic day 12 in the peripheral nerves and their somata. Intense NCS-1 IR was expressed in ascending and descending tracts in the white matter during the late prenatal period, which gradually decreased to the faint level during postnatal development. Intense NCS-1 IR was colocalized with growth associated protein (GAP)-43 IR in the marginal zone and with the glutamate-aspartate transporter (GLAST) IR in the radial processes traversing the marginal zone. In the adult rat white matter, radially oriented astrocytes and astrocytes in the glia limitans were double-labeled for NCS-1 and glial fibrillary acidic protein (GFAP), whereas small dots on finger-like dendritic projections were double-labeled for NCS-1 and synaptophysin. In the developing gray matter, the NCS-1 IR appeared at embryonic day 12 and gradually increased in the neuronal somata and neuropil, reaching a plateau after the end of the 4th postnatal week. The small dots in neuropil were colabeled for NCS-1 and GFAP or NCS-1 and synaptophysin in the adult rat gray matter. These results strongly suggest that NCS-1 is involved in axogenesis and synaptogenesis in the developing rat spinal cord. NCS-1 can serve as a Ca(2+)-sensor not only in neurons but also in radial glial cells or even in radially oriented astrocytes in the adult rat spinal cord.
|Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species. |
Liang, Xiquan, et al.
J. Biol. Chem., 277: 33032-40 (2002) 2002
GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.
|Collateral sprouting mechanism after end-to-side nerve repair in the rat. |
T Yamauchi, M Maeda, S Tamai, M Tamai, H Yajima, Y Takakura, S Haga, H Yamamoto
Medical electron microscopy : official journal of the Clinical Electron Microscopy Society of Japan 33 151-6 2000
The collateral sprouting mechanisms of axons from an uninjured donor nerve after end-to-side nerve repair was investigated in motor nerves of rats, with special reference to the neurotrophins related to nerve regeneration. In addition, growth cone formation at the tip of the regenerating nerve was examined. A transected medial gastrocnemius nerve (MGN) was transferred to the side of an intact lateral gastrocnemius nerve (LGN) using a Y-shaped silicone tube. At 3, 7, or 14 days later, the MGN with the LGN was transected and was stained immunohistologically. Expression of neurotrophin-3 (NT-3) and Trk C (receptor of NT-3) was most significantly observed 3 days postoperatively around the site of coaptation. Brain-derived neurotrophic factor (BDNF) and Trk B (receptor of BDNF) was weakly detected at the coaptation site 3 days after-operation. Growth-associated protein 43 (GAP-43), which is a marker of growth cone formation, was observed at the site of coaptation in the LGN 7 days postoperatively and in the MGN at the site of coaptation at 14 days. We concluded that motor nerve regeneration due to collateral sprouting of axons after end-to-side nerve repair is possible. We thus demonstrated the involvement of at least one neurotrophin, NT-3, in the process of collateral sprouting of motor nerves.
|5'-Nucleotidase activity indicates sites of synaptic plasticity and reactive synaptogenesis in the human brain. |
Lie, A A, et al.
J. Neuropathol. Exp. Neurol., 58: 451-8 (1999) 1999
The localization and morphological assessment of plastic or newly formed synapses in the human brain remains difficult due to the lack of specific markers. The ectoenzyme 5'-nucleotidase may represent a useful marker of these structures, since in adult rodents synaptic 5'-nucleotidase activity is restricted to sites of spontaneous synaptic turnover and induced reactive synaptogenesis. However, it is unclear to what extent synaptic 5'-nucleotidase activity occurs in the normal human brain, and whether reactive synaptogenesis, as seen e.g. in temporal lobe epilepsy (TLE), is associated with this ectoenzyme. Therefore, we have investigated the histochemical distribution of 5'-nucleotidase in hippocampal control specimens (n = 3) and in the hippocampus of TLE patients (n = 13). In controls, 5'-nucleotidase activity was present in the dentate gyrus molecular layer (DG-ML) and the mossy fiber termination field within the CA4 and CA3 subfields. Compared with controls, TLE specimens revealed markedly increased 5'-nucleotidase labeling in the DG-ML, implying TLE-associated reactive synaptogenesis in this hippocampal region. In contrast to GAP-43, synaptophysin, and dynorphin A, synaptic 5'-nucleotidase activity may serve as a potential specific indicator of plastic synapses or newly formed terminals in the human brain and prove useful for the study of diseases involving aberrant sprouting or altered synaptic plasticity.
|Fate of GAP-43 in ascending spinal axons of DRG neurons after peripheral nerve injury: delayed accumulation and correlation with regenerative potential. |
Schreyer, D J and Skene, J H
J. Neurosci., 11: 3738-51 (1991) 1991
Proteins characteristic of growing axons often fail to be induced or transported along axons that have been interrupted far from their cell bodies in the adult mammalian CNS. Here, we inquire whether long axons in the mammalian CNS can support efficient axonal transport and deposition of one such protein, GAP-43, when the protein is induced in neuron cell bodies. We have used immunocytochemistry to follow the fate of GAP-43 in dorsal column axons ascending the rat spinal cord from dorsal column axons ascending the rat spinal cord from dorsal root ganglion (DRG) neurons, after synthesis of the protein is induced in these cells by peripheral nerve injury. Sciatic nerve lesions do lead to an accumulation of GAP-43 in dorsal column axons derived from the lumbar DRG. However, in distal segments of these CNS axons, accumulation of GAP-43 is apparent only after a delay of 1-2 weeks, in contrast to its rapid accumulation in axon segments within the PNS environment, suggesting that deposition and stabilization of GAP-43 can be limited by local, posttranslational regulation. GAP-43 immunoreactivity subsides to control levels within 8 weeks after crush lesions that permit peripheral axon regeneration, but remains robust 8 weeks after resection lesions that prevent peripheral regeneration. Accumulation of GAP-43 in cervical dorsal column axons after peripheral nerve injury is closely correlated with the ability of these axons to respond to local cues capable of eliciting axon growth (Richardson and Verge, 1986).
|Changes in the distribution of GAP-43 during the development of neuronal polarity. |
Goslin, K, et al.
J. Neurosci., 10: 588-602 (1990) 1990
GAP-43, a neuron specific growth-associated protein, is selectively distributed to the axonal domain in developing neurons; it is absent from dendrites and their growth cones. Using immunofluorescence microscopy, we have further examined the distribution of GAP-43 during the development of hippocampal neurons in culture, in order to determine when this polarized distribution arises. Cultured hippocampal neurons initially extend several short processes which have the potential to become either axons or dendrites. At this stage, before the morphological expression of polarity, GAP-43 is concentrated in the growth cones of these processes but is distributed more or less equally among them. Polarity becomes established when one of these processes elongates to become the axon. At the earliest stage when the emerging axon can be identified, GAP-43 is preferentially concentrated in its growth cone. During the next few days, as the remaining processes take on dendritic properties, they lose their residual GAP-43 immunoreactivity. Throughout development, GAP-43 remains highly concentrated in the axonal growth cone, but the concentration of GAP-43 in the axon shaft increases, beginning near the growth cone and progressing proximally until GAP-43 is uniformly distributed along the entire axon. At all stages of development, GAP-43 is also concentrated in the region of the Golgi apparatus. These results suggest that the selective sorting of at least one membrane protein into the axon coincides with the morphological expression of polarity. These results also raise the possibility that GAP-43 may play an important role in the early phases of axonal outgrowth, by which the functional polarity of neurons is established.
|Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. |
Skene, J H and Virág, I
J. Cell Biol., 108: 613-24 (1989) 1989
Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP-43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent-enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.
|Anti-Growth Associated Protein 43, clone 9-1E12 - Data Sheet|