The SCG10-related gene family in the developing rat retina: persistent expression of SCLIP and stathmin in mature ganglion cell layer

Neuronal growth-associated proteins (GAPs), such as GAP-43 and SCG10, are thought to play crucial roles in both axonal and dendritic outgrowth during neural development and regeneration, although the underlying mechanisms remain largely unknown. The recent finding that SCG10 is a microtubule regulator and also the identification of RB3 and SCLIP as two new SCG10-related members prompted us to investigate the roles of SCG10-related family in neural development, using the retina as a model system. We determined the temporal expression and the spatial distribution of SCG10-related mRNAs in the developing rat retina. Semiquantitative analysis by RT-PCR revealed that in prenatal retina, levels of SCG10 and stathmin mRNAs were higher than those of RB3 and SCLIP. In the postnatal retina, the level of SCLIP increased, whereas the level of RB3 remained low. In situ hybridization revealed that GAP-43 and all of the SCG10-related family mRNAs were present in the retinal ganglion cells (RGCs) at all stages of retinal development, and that stathmin mRNA was present in mitotic neuroblastic cells. Differential expression of SCG10 and other members of the family became more evident as retinal development proceeded; SCG10 and RB3 expression were relatively specific in the RGCs and amacrine cells, whereas SCLIP was also evident in bipolar and horizontal cells. Stathmin mRNA was highly expressed both in the RGCs and other interneurons. These results indicate that multiple SCG10-related proteins are expressed in single neurons including RGCs, and suggest that these nGAPs play similar but distinct roles in differentiation and functional maintenance of retinal neurons.     

Taxol and tau overexpression induced calpain-dependent degradation of the microtubule-destabilizing protein SCG10

Microtubule-stabilizing and -destabilizing proteins play a crucial role in regulating the dynamic instability of microtubules during neuronal development and synaptic transmission. The microtubule-destabilizing protein SCG10 is a neuron-specific protein implicated in neurite outgrowth. The SCG10 protein is significantly reduced in mature neurons, suggesting that its expression is developmentally regulated. In contrast, the microtubule-stabilizing protein tau is expressed in mature neurons and its function is essential for the maintenance of neuronal polarity and neuronal survival. Thus, the establishment and maintenance of neuronal polarity may down-regulate the protein level/function of SCG10. In this report, we show that treatment of PC12 cells and neuroblastoma cells with the microtubule-stabilizing drug Taxol induced a rapid degradation of the SCG10 protein. Consistently, overexpression of tau protein in neuroblastoma cells also induced a reduction in SCG10 protein levels. Calpain inhibitor MDL-28170, but not caspase inhibitors, blocked a significant decrease in SCG10 protein levels. Collectively, these results indicate that tau overexpression and Taxol treatment induced a calpain-dependent degradation of the microtubule-destabilizing protein SCG10. The results provide evidence for the existence of an intracellular mechanism involved in the regulation of SCG10 upon microtubule stabilization.     

Expression of stathmin and SCG10 proteins in the olfactory neurogenesis during development and after lesion in the adulthood

Stathmin and SCG10 belong to a family of phosphoproteins associated to cell proliferation and differentiation. In the present study, we have analyzed immunocytochemically the distribution of these proteins during neurogenesis in the mouse olfactory system, from midgestation to adulthood. Data show that already at embryonic day 12, stathmin and SCG10 immunoreactivities were present in the olfactory and vomeronasal neurons, and their number increased greatly, colocalizing with neuronal specific tubulin, a marker of immature neurons. Later on up to adulthood, the distribution of stathmin and SCG10 became progressively restricted to a few immature receptor and chemosensory neurons. Significantly, in the olfactory epithelium, stathmin was seen in immature neurons and also in basal cells representing precursors of neuronal elements. Interestingly, before birth stathmin and SCG10 immunopositive cells were seen outside the olfactory epithelium, seemingly migrating toward the olfactory bulb. After regeneration in the adult following peripheral lesion of the olfactory epithelium, stathmin and SCG10 were again strongly expressed and generally colocalized with neuronal specific tubulin immunoreactivity. Overall these results indicate that stathmin and SCG10 are expressed in immature olfactory neurons as well as in the migrating cells generated from the olfactory epithelium, supporting the role of these proteins in neurogenesis and cell migration.     

Abnormal expression of stathmin 1 in brain tissue of patients with intractable temporal lobe epilepsy and a rat model

Microtubule dynamics have been shown to contribute to neurite outgrowth, branching, and guidance. Stathmin 1 is a potent microtubule-destabilizing factor that is involved in the regulation of microtubule dynamics and plays an essential role in neurite elongation and synaptic plasticity. Here, we investigate the expression of stathmin 1 in the brain tissues of patients with intractable temporal lobe epilepsy (TLE) and experimental animals using immunohistochemistry, immunofluorescence and western blotting. We obtained 32 temporal neocortex tissue samples from patients with intractable TLE and 12 histologically normal temporal lobe tissues as controls. In addition, 48 Sprague Dawley rats were randomly divided into six groups, including one control group and five groups with epilepsy induced by lithium chloride-pilocarpine. Hippocampal and temporal lobe tissues were obtained from control and epileptic rats on Days 1, 7, 14, 30, and 60 after kindling. Stathmin 1 was mainly expressed in the neuronal membrane and cytoplasm in the human controls, and its expression levels were significantly higher in patients with intractable TLE. Moreover, stathmin 1 was also expressed in the neurons of both the control and the experimental rats. Stathmin 1 expression was decreased in the experimental animals from 1 to 14 days postseizure and then significantly increased at Days 30 and 60 compared with the control group. Many protruding neuronal processes were observed in the TLE patients and in the chronic stage epileptic rats. These data suggest that stathmin 1 may participate in the abnormal network reorganization of synapses and contribute to the pathogenesis of TLE.     

Prenatal GABAB1 and GABAB2 receptors: cellular and subcellular organelle localization in early fetal rat cortical neurons

Gamma-aminobutyric acid (GABA)(B) receptors appear to influence developmental events, depending on whether they are found at a synapse or in extrasynaptic areas. Little, if anything, is known as to the cellular and subcellular localization of GABA(B1) and GABA(B2) receptors during early fetal development. We used Western blots, immunohistochemistry, and postembedding immunoelectronmicroscopy to investigate fetal rat brain expression and distribution of these receptor proteins. GABA(B1) is expressed as early as gestational day (GD) 11.5 and 12.5, with immunoreactivity found in the all neuroepithelium, and a high expression in the mantel zone and the cortical area's plate; no immunolabeling for GABA(B2) receptor was observed. Our immunogold studies define a pattern of early GABA(B1) receptor protein in dendrite processes, endoplasmic reticulum, and axon terminals of the cortical neuroepithelium on GD 11.5. On GD 12.5, GABA(B1) receptor immunogold was found in dendrite processes, spines and tree, axon terminals, mitochondria, and intracellular organelles of the cortical neuroepithelium. No synapse formation was apparent as no synaptophysin could be found on either GD 11.5 or 12.5. We suggest that GABA(B1) has a functional role in the early fetal brain during neuronal proliferation and migration, and that it is different from the established functional GABA(B) receptor.     

Protein kinase C gamma interneurons in the rat medullary dorsal horn: Distribution and synaptic inputs to these neurons,and subcellular localization of the enzyme

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (II_i) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina II_i PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A‐ and C‐fibers low threshold mechanoreceptors (LTMRs) terminate in lamina II_i raise the general issue of synaptic inputs to lamina II_i PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ‐immunoreactivity is mostly restricted to interneurons in lamina II_i of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ‐immunoreactive interneurons are asymmetric (likely excitatory). PKCγ‐immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3‐immunoreactive bouton, specific to C‐LTMRs, on PKCγ‐immunoreactive interneurons. PKCγ‐immunoreactive interneurons contain GABA_Aergic and glycinergic receptors. At the subcellular level, PKCγ‐immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ‐immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia. J. Comp. Neurol. 522:393–413, 2014. © 2013 Wiley Periodicals, Inc.     

Effect of taxoid and nontaxoid site microtubule‐stabilizing agents on axonal transport of mitochondria in untransfected and ECFP‐htau40‐transfected rat cortical neurons in culture

An important aspect of synaptic plasticity in the brain is axonal transport of essential components such as mitochondria from the soma to the synapse. For uninterrupted transport of cellular cargo down the axon, functional microtubules are required. Altered microtubule dynamics induced by changes in expression of microtubule‐associated tau protein affects normal microtubule function and interferes with axonal transport. Here we investigate the effects of the nontaxoid‐binding‐site microtubule‐stabilizing agents peloruside A (PelA) and laulimalide, compared with the taxoid‐site‐binding agents paclitaxel (Ptx) and ixabepilone, on axonal transport of mitochondria in 1‐day‐old rat pup cerebral cortical neuron cultures. The differences in effects of these two types of compound on mitochondrial trafficking were specifically compared under conditions of excess tau expression. PelA and laulimalide had no adverse effects on their own on mitochondrial transport compared with Ptx and ixabepilone, which inhibited mitochondrial run length at higher concentrations. PelA, like Ptx, was able to partially reverse the blocked mitochondrial transport seen in ECFP‐htau40‐overexpressing neurons, although at higher concentrations of microtubule‐stabilizing agent, the PelA response was improved over the Ptx response. These results support a neuroprotective effect of microtubule stabilization in maintaining axonal transport in neurons overexpressing tau protein and may be beneficial in reducing the severity of neurodegenerative diseases such as Alzheimer's disease. © 2014 Wiley Periodicals, Inc.     

骨桥蛋白与整合素β3在脑胶质瘤中的表达及其意义

目的探讨骨桥蛋白（OPN）、整合素β3与胶质瘤恶性程度的关系,及OPN与整合素β3之间的相关性.方法采用免疫组化染色、逆转录-聚合酶链反应（RT-PCR）及免疫印迹法（Western B1ot）等方法检测正常人脑组织和胶质瘤组织中OPN、整合素β3的mRNA及蛋白表达.结果正常脑组织中无OPN、整合素β3表达,而胶质瘤细胞膜、细胞质及血管内皮细胞中有表达.随着脑胶质瘤病理分级的增高,OPN与整合素β3的表达显著性增高（P＜0.05）.OPN与整合素β3的mRNA（r=0.990,P＜0.05）及蛋白（=0.943,P＜0.05）表达均呈正相关.结论OPN与整合素β3在人脑胶质瘤组织中的表达强度与其恶性程度相关,且OPN和整合素β3的表达之间有内在联系.     

Cellular and subcellular localization of estrogen and progestin receptor immunoreactivities in the mouse hippocampus

Estrogen receptor‐α (ERα), estrogen receptor‐β (ERβ), and progestin receptor (PR) immunoreactivities are localized to extranuclear sites in the rat hippocampal formation. Because rats and mice respond differently to estradiol treatment at a cellular level, the present study examined the distribution of ovarian hormone receptors in the dorsal hippocampal formation of mice. For this, antibodies to ERα, ERβ, and PR were localized by light and electron immunomicroscopy in male and female mice across the estrous cycle. Light microscopic examination of the mouse hippocampal formation showed sparse nuclear ERα and PR immunoreactivity (‐ir) most prominently in the CA1 region and diffuse ERβ‐ir primarily in the CA1 pyramidal cell layer as well as in a few interneurons. Ultrastructural analysis additionally revealed discrete extranuclear ERα‐, ERβ‐, and PR‐ir in neuronal and glial profiles throughout the hippocampal formation. Although extranuclear profiles were detected in all animal groups examined, the amount and types of profiles varied with sex and estrous cycle phase. ERα‐ir was highest in diestrus females, particularly in dendritic spines, axons, and glia. Similarly, ERβ‐ir was highest in estrus and diestrus females, mainly in dendritic spines and glia. Conversely, PR‐ir was highest during proestrus, mostly in axons. Except for very low levels of extranuclear ERβ‐ir in mossy fiber terminals in mice, the labeling patterns in the mice for all three antibodies were similar to the ultrastructural labeling found previously in rats, suggesting that regulation of these receptors is well conserved across the two species. J. Comp. Neurol. 518:2729–2743, 2010. © 2010 Wiley‐Liss, Inc.     

Differential subcellular localization of phosphorylated neurofilament and tau proteins in degenerating neurons of the human entorhinal cortex

A panel of novel monoclonal antibodies was tested on the human entorhinal cortex for the recognition of age- and disease-related changes of neurofilament proteins (NF). Several antibodies identified phosphorylated NF-H subunit, which occurred preferentially in those aged between 60 and 80 years and were localized in degenerating neurons. Such neurons also contained neurofibrillary tangles, but neurofilament aggregates did not co-localize with tangles, nor did the quantity nor the number of NF-positive neurons correlate with the severity of Alzheimer's disease. This points to a susceptibility of NF in a subset of neurons for phosphorylation- and metabolically related morphological changes during neurodegeneration.     

GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members

The tubby gene family consists of four members, TUB, TULP1, TULP2 and TULP3, with unknown function. However, a splice junction mutation within the mouse tub gene leads to retinal and cochlear degeneration, as well as maturity onset obesity and insulin resistance. Mutations within human TULP1 have also been shown to co-segregate in several cases of autosomal recessive retinitis pigmentosa (RP) and TULP1 deficiency in mice leads to retinal degeneration. The primary amino acid sequences of the tubby family members do not predict a likely biochemical function. As a first step in defining their function, we present a detailed characterization of the cellular and subcellular localization of the human (TUB) and mouse (tub) homologous gene products. We report the isolation of TUB splice variants which have different subcellular localizations (nuclear versus cytoplasmic) and which define a nuclear localization signal. In addition, using green fluorescent protein (GFP) tags, we observe a nuclear localization for TULP1, similar to TUB splicing forms TUB 561 and TUB 506. Finally, we report tubby expression in mouse brain by in situ hybridization and by immunohistochemistry with polyclonal antibodies. Protein was found in both the hypothalamic satiety centers and in a variety of other CNS structures including the cortex, cerebellum, olfactory bulb and hippocampus. Both nuclear and cytoplasmic signals were detected with a series of independently generated polyclonal antibodies, consistent with the presence of multiple alternatively spliced isoforms within the CNS.     

Expression and subcellular localization of Ror tyrosine kinase receptors are developmentally regulated in cultured hippocampal neurons

Ror1 and Ror2 are two novel receptor tyrosine kinases that have been implicated in neuronal differentiation in Caenorhabditis elegans. As a first step toward elucidating their role in the mammalian brain, we analyzed their expression and localization patterns in hippocampal neurons. Our results showed that both receptors are expressed from early stages of development and that their protein levels peak during periods of active synapse formation. Immunocytochemical analysis indicated that Ror1 and Ror2 are highly concentrated in the growth cones of immature neurons and are present throughout the somatodendritic compartment of mature hippocampal cells. Further analysis indicated that they are present not only in the cell membrane but also in Triton- and saponin-insoluble fractions, suggesting that they may be associated with both the cytoskeleton and membrane-bound organelles. Taken collectively, our results suggest that Ror1 and Ror2 might play a role during early stages of development in mammalian central neurons.     

Differential distribution of stathmin and SCG10 in developing neurons in culture

The neuron-specific protein SCG10 and the ubiquitous protein stathmin are two members of a family of microtubule-destabilizing factors that may regulate microtubule dynamics in response to extracellular signals. To gain insight into the function of these proteins in the nervous system, we have compared their intracellular distribution in cortical neurons developing in culture. We have used double-immunofluorescence microscopy with specific antibodies for stathmin and SCG10 in combination with antibodies for axonal, microtubule, and synaptic marker proteins. Stathmin and SCG10 were coexpressed in individual neurons. While both proteins were highly expressed in developing cultures during differentiation, their subcellular localization was strikingly different. Stathmin showed a cytosolic distribution, mainly in cell bodies, whereas SCG10 strongly labeled the growth cones of axons and dendrites. During neurite outgrowth, SCG10 appeared as a single concentrated spot in a region of the growth cone where the microtubules are known to be particularly dynamic. Disassembly of labile microtubules by nocodazole caused a dispersal of the SCG10 staining into punctate structures, indicating that its subcellular localization is microtubule-dependent. Upon maturation and synapse formation, the levels of both stathmin and SCG10 decreased to become undetectable. These observations demonstrate that the expression of both proteins is associated with neurite outgrowth and suggest that they perform their roles in this process in distinct subcellular compartments. J. Neurosci. Res. 50:1000–1009, 1997. © 1997 Wiley-Liss, Inc.     

Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons

Immunoblot analysis, using antibodies against distinct N-methyl-d -aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.     

磷酸化Rb蛋白在正常成年大鼠与脑缺血大鼠神经元内的表达变化

目的观察正常成年大鼠与局灶性脑缺血大鼠神经元内磷酸化Rb蛋白(p-Rb)的表达及变化特点。方法应用免疫荧光组织化学染色方法观察成年大鼠不同脑区神经元内p-Rb表达特点,比较大鼠大脑中动脉闭塞(MCAO)后不同时间点(12h、1d、3d、7d)缺血半暗带内神经元p-Rb的变化特点。结果成年大鼠神经元内p-Rb主要在细胞核内表达;与对照组相比,MCAO再灌注后各时间点p-Rb免疫反应性均增强;再灌注1d,缺血半暗带内部分神经元p-Rb表达由细胞核转移到细胞浆。结论成熟神经元内p-Rb在细胞核内表达可能对于维持神经元的有丝分裂后状态有一定作用,当其发生核浆转移提示神经元细胞周期调控机制紊乱,可能再进入细胞周期。     

Antibody to the extracellular domain of the low affinity NGF receptor stimulates p75(NGFR)-mediated apoptosis in cultured sympathetic neurons

Recent evidence has established a role for p75(NGFR) in developmentally regulated neuronal cell death. Although cell death due to NGF withdrawal is a well described, apoptosis in sympathetic neurons through stimulation of p75(NGFR) has not been clearly demonstrated. We have found that an antibody directed against the extracellular domain of murine p75(NGFR) profoundly effects the survival of short-term cultures of sympathetic neurons. Rat superior cervical ganglion neurons grown in the presence of NGF and treated with the bioactive antibody (9651) display a dose-dependent increase in cell death. This effect was independent of NGF concentration and partially reversed by either depolarizing stimuli or forskolin. The response to 9651 seems to act directly through a p75(NGFR)-mediated pathway and not by disturbing p75(NGFR)/TrkA interactions. Moreover, the kinetics of antibody stimulated cell death was more rapid than the cell death resulting from removal of NGF and treatment with CNTF failed to promote neuronal survival in the presence of 9651. Initiation of cell death is often associated with decreased NFkappaB activity, whereas survival or rescue correlates with increased NFkappaB. Increases in NFkappaB, however, have been observed in neurons in several diseases and late in apoptosis in differentiated PC12 cells. Time course studies revealed a rapid decrease in NFkappaB activity and a slight, but persistent increase in binding that correlated with decline in cell numbers 3 hr after treatment. These results suggest the cell death program is initiated shortly after antibody activation of p75(NGFR) and a subpopulation of cells may remain susceptible to rescue.     

GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons

Our objective was to determine whether GABAergic and cholinergic basal forebrain neurons project to the neocortex. The retrograde connectivity marker wheat germ agglutinin lectin-bound horseradish peroxidase was injected into the neocortex of adult cats. Histo- and immunohistochemical methods were combined to label sequentially connectivity and transmitter markers (glutamic acid decarboxylase; choline acetyltransferase) in forebrain neurons. The labels of each marker were identified by correlative light and electron microscopy. Two principal types of doubly labeled neurons were demonstrated. The connectivity marker was colocalized with glutamic acid decarboxylase or choline acetyltransferase. The neurons were located in the basal forebrain. Their ultrastructural, cellular, and regional organization supported 2 conclusions. (1) GABAergic basal forebrain neurons project to the neocortex. This is important new morphological evidence for the origin of inhibitory neocortical afferents from a subcortical brain site. (2) The GABAergic and cholinergic basal forebrain neurons projecting to the neocortex exhibit remarkable structural similarities. The transmitter diversity of these intertwined neocortical afferents may be significant for the pathology and treatment of human neurological disorders such as Alzheimer's disease.