The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.     

JNK1 phosphorylation of SCG10 determines microtubule dynamics and axodendritic length

c-Jun NH(2)-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3' interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1-/- cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site-phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.     

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-beta (SAPKbeta) binds and phosphorylates the neuronal microtubule regulator SCG10.

The neuronal growth-associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule-destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine-threonine-directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen-activated protein (MAP) kinases, c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKbeta. JNK3/SAPKbeta phosphorylation occurs at Ser-62 and Ser-73, residues that result in reduced microtubule-destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKbeta activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.     

Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth

Calmyrin1 (CaMy1) is an EF-hand Ca²⁺-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca²⁺-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca²⁺ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Stathmin and its phosphoprotein family: general properties, biochemical and functional interaction with tubulin

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.     

Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.     

Amyloid beta oligomerization is induced by brain lipid rafts

Detergent-resistant lipid rafts are required for the generation of Abeta as they concentrate not only amyloid precursor protein (APP), but also the beta- and gamma-secretase that convert APP to Abeta. Recently, Abeta has been shown to be oligomerized, which results in neuronal cytotoxicity and synaptic failure. In this study, we have demonstrated that Abeta oligomers appeared immediately after the incubation of Abeta with lipid rafts isolated from the brain tissues of rats, and were converted into few Abeta fibrils, even after longer periods of incubation. The oligomerization of Abeta was not abolished after the brain lipid rafts were treated with heat, or with protease K, implying that the lipid raft proteins were determined not to be prerequisites for Abeta oligomerization. The cholesterol present in the lipid rafts might not be essential to Abeta oligomerization because Abeta oligomerization was not prevented after the cholesterol was removed from the lipid rafts with methyl-beta-cyclodextrin (MbetaCD). The Abeta oligomerization was accelerated by the application of lipid rafts isolated from ganglioside-rich cells, C2C12 cells, whereas this was not observed with the lipid rafts isolated from ganglioside-poor cells SK-N-MC and HeLa cells. In addition, lipid raft-induced Abeta oligomerization was shown to be inhibited in CHO-K1 cells which were defective with regard to ganglioside biosynthesis. This indicates that Abeta oligomerization requires gangliosides that are enriched in the lipid rafts.     

SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons

Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.     

Differential targeting of Shaker-like potassium channels to lipid rafts

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitric-oxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltage-gated K(+) channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K(+) channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.     

Tubulin-Associated Calmodulin-Dependent Kinase: Evidence for an Endogenous Complex of Tubulin with a Calcium-Calmodulin-Dependent Kinase

A Ca2+ -calmodulin kinase that phosphorylates tubulin and microtubule-associated proteins as major substrates has been purified and characterized from brain cytoplasm. It is important to determine if cytoskeletal proteins are major natural substrates for this kinase system. This report demonstrates that a significant fraction of brain cytosolic calmodulin-dependent kinase activity exists in tight association with tubulin in the form of a stable complex. The tubulin-calmodulin kinase complex displayed an apparent molecular weight on gel filtration of approximately 1.8 X 10(6) daltons. The specific activity of tubulin kinase in the complex was enriched over 20-fold in comparison with brain cytosol. Although purified tubulin alone did not adhere to a calmodulin column, the tubulin associated with the calmodulin kinase complex did bind specifically to the calmodulin affinity resin. The kinase activity was shown to be tightly associated in complex with tubulin by (1) copurification, (2) isolation on gel filtration chromatography, (3) isolation on ion-exchange chromatography, and (4) binding to calmodulin. The kinase complexed with tubulin was identical to the previously purified kinase as judged by several criteria including (1) subunit molecular weights, (2) isoelectric points, (3) autophosphorylation characteristics, (4) calmodulin binding properties, (5) kinetic parameters of tubulin phosphorylation, (6) phosphoamino acid phosphorylation sites on alpha- and beta-tubulin, and (7) identical subunit 125I-tryptic peptide maps. The results indicate that a significant fraction of this previously purified calmodulin kinase is endogenously associated with tubulin in brain cytoplasm and may play a role in mediating some of the effects of calcium on neuronal function.     

Miswiring the brain: Δ9‐tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin‐2 degradation pathway

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ⁹‐tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor‐driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB₁ cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC‐sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin‐2, a microtubule‐binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis‐exposed fetuses, defining SCG10 as the first cannabis‐driven molecular effector in the developing cerebrum. CB₁ cannabinoid receptor activation recruits c‐Jun N‐terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.     

The b1 isoform of protocadherin-gamma (Pcdhgamma) interacts with the microtubule-destabilizing protein SCG10

Due to their structural characteristics and their diversity, the 22 members of the protocadherin-gamma (Pcdhgamma) family have been suggested to contribute to the establishment of specific connections in the nervous system. Here, we focus on a single isoform, Pcdhgamma-b1. Its expression is found in different brain regions and in developing spinal cord it is restricted to scattered cells, whereas all cells are labeled using an antibody that recognizes all Pcdhgamma isoforms. As a first step to understanding the signaling mechanisms downstream of Pcdhgamma, we identify the microtubule-destabilizing protein SCG10 as a cytoplasmic interactor for Pcdhgamma-b1 and other isoforms of the Pcdhgamma-b subfamily, and show that SCG10 and Pcdhgamma-b1 are found together in certain neuronal growth cones.     

RGS6 interacts with SCG10 and promotes neuronal differentiation. Role of the G gamma subunit-like (GGL) domain of RGS6

RGS proteins comprise a large family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. RGS6 is a member of the R7 RGS protein subfamily endowed with DEP (disheveled, Egl-10, pleckstrin) and GGL (G protein gamma subunit-like) domains in addition to the RGS domain present in all RGS proteins. RGS6 exists in multiple splice variant forms with identical RGS domains but possessing complete or incomplete GGL domains and distinct N- and C-terminal domains. Here we report that RGS6 interacts with SCG10, a neuronal growth-associated protein. Using yeast two-hybrid analysis to map protein interaction domains, we identified the GGL domain of RGS6 as the SCG10-interacting region and the stathmin domain of SCG10 as the RGS6-interacting region. Pull-down studies in COS-7 cells expressing SCG10 and RGS6 splice variants revealed that SCG10 co-precipitated RGS6 proteins with complete GGL domains but not those with incomplete GGL domains, and vice versa. Expression of SCG10-interacting forms of RGS6 with SCG10 in PC12 or COS-7 cells resulted in co-localization of both proteins. RGS6 potentiated the ability of SCG10 to disrupt microtubule organization in PC12 and COS-7 cells. Furthermore, expression of SCG10 and RGS6 each enhanced NGF-induced PC12 cell differentiation, and co-expression of SCG10 with RGS6 produced synergistic effects on NGF-induced PC12 differentiation. These effects of RGS6 on microtubules and neuronal differentiation were observed only with RGS6 proteins with complete GGL domains. Mutation of a critical residue required for interaction of RGS proteins with G proteins did not affect the ability of RGS6 to induce neuronal differentiation. These findings identify SCG10 as a binding partner for the GGL domain of RGS6 and provide the first evidence for regulatory effects of an RGS protein on neuronal differentiation. Our results suggest that RGS6 induces neuronal differentiation by a novel mechanism involving interaction of SCG10 with its GGL domain and independent of RGS6 interactions with heterotrimeric G proteins.     

SCLIP: A Novel SCG10-Like Protein of the Stathmin Family Expressed in the Nervous System

Abstract: Stathmin is a cytosolic phosphoprotein previously described as a ubiquitous relay integrating various signaling pathways. It is the generic element of a protein family in mammals as in Xenopus , including SCG10, a neuron-specific, growth-associated protein, and RB3/XB3, related to the expression of differentiated functions of mature cells of the nervous system. In an extensive search for other members of the stathmin family, we identified cDNAs coding for two novel stathmin-related proteins: (a) a cDNA from a rat striatum cDNA library codes for RB3", a splice variant of RB3, with an additional basic domain in its N-terminal region; and (b) another cDNA identified through a systematic search in EST databases codes for a novel protein, SCLIP, for "SCG10-like protein," displaying 70% identity with SCG10 and sharing the same domain organization, with an N-terminal domain likely involved in membrane attachment and a C-terminal stathmin-like domain. Northern blot analysis as well as in situ hybridization on 14-day rat embryos showed that SCLIP mRNA is expressed only in neural structures. SCLIP mRNA is expressed at comparable levels in neonatal and adult rat brain, suggesting a potential role not only in the acquisition, but also in the expression of differentiated neuronal functions.     

Stathmin family proteins display specific molecular and tubulin binding properties

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.     

The interaction of RGSZ1 with SCG10 attenuates the ability of SCG10 to promote microtubule disassembly

RGS proteins (regulators of G protein signaling) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins. Initially characterized as GTPase-activating proteins for Galpha subunits, recent data have implied additional functions for RGS proteins. We previously identified an RGS protein (termed RGSZ1) whose expression is quite specific to neuronal tissue (Glick, J. L., Meigs, T. E., Miron, A., and Casey, P. J. (1998) J. Biol. Chem. 273, 26008-26013). In a continuing effort to understand the role of RGSZ1 in cellular signaling, the yeast two-hybrid system was employed to identify potential effector proteins of RGSZ1. The microtubule-destabilizing protein SCG10 (superior cervical ganglia, neural specific 10) was found to directly interact with RGSZ1 in the yeast system, and this interaction was further verified using direct binding assays. Treatment of PC12 cells with nerve growth factor resulted in Golgi-specific distribution of SCG10. A green fluorescent protein-tagged variant of RGSZ1 translocated to the Golgi complex upon treatment of PC12 cells with nerve growth factor, providing evidence that RGSZ1 and SCG10 interact in cells as well as in vitro. Analysis of in vitro microtubule polymerization/depolymerization showed that binding of RGSZ1 to SCG10 effectively blocked the ability of SCG10 to induce microtubule disassembly as determined by both turbidimetric and microscopy-based assays. These results identify a novel connection between RGS proteins and the cytoskeletal network that points to a broader role than previously envisioned for RGS proteins in regulating biological processes.     

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases.     

Role of the microtubule destabilizing proteins SCG10 and stathmin in neuronal growth

The related proteins SCG10 and stathmin are highly expressed in the developing nervous system. Recently it was discovered that they are potent microtubule destabilizing factors. While stathmin is expressed in a variety of cell types and shows a cytosolic distribution, SCG10 is neuron-specific and membrane-associated. It contains an N-terminal targeting sequence that mediates its transport to the growing tips of axons and dendrites. SCG10 accumulates in the central domain of the growth cone, a region that also contains highly dynamic microtubules. These dynamic microtubules are known to be important for growth cone advance and responses to guidance cues. Because overexpression of SCG10 strongly enhances neurite outgrowth, SCG10 appears to be an important factor for the dynamic assembly and disassembly of growth cone microtubules during axonal elongation. Phosphorylation negatively regulates the microtubule destabilizing activity of SCG10 and stathmin, suggesting that these proteins may link extracellular signals to the rearrangement of the neuronal cytoskeleton. A role for these proteins in axonal elongation is also supported by their growth-associated expression pattern in nervous system development as well as during neuronal regeneration.     

Tubulin is a neuronal target of autoantibodies in Sydenham's chorea

Sydenham's chorea is a CNS disorder and sequela of group A streptococcal infection where deposition of Abs in brain may result in movement and neuropsychiatric abnormalities. We studied human mAbs 24.3.1, 31.1.1, and 37.2.1 derived from chorea and selected for cross-reactivity with group A streptococci and brain Ags. Our novel findings reveal that Sydenham's chorea mAbs target a 55-kDa brain protein with an N-terminal amino acid sequence of MREIVHLQ corresponding to beta-tubulin. Chorea mAb specificity for purified brain tubulin was confirmed in ELISA and Western immunoblot, and significant levels of anti-tubulin IgG were found in acute chorea sera and cerebrospinal fluid. Lysoganglioside G(M1) inhibited binding of chorea mAbs to tubulin and mAb reactivity with human caudate and putamen brain sections was blocked by anti-tubulin mAb. The chorea mAbs labeled both intra- and extracellular Ags of a neuronal cell line providing evidence suggesting mimicry between intracellular brain protein tubulin and extracellular lysoganglioside. In addition, chorea mAb 24.3.1 and acute chorea sera induced calcium/calmodulin-dependent protein kinase II activity in human neuronal cells. Nucleotide sequence analysis of the chorea mAb V(H) genes revealed that mAb 24.3.1 V(H) gene was encoded by the V(H)1 germline gene family which encodes other anti-ganglioside V(H) genes associated with motor neuropathies. mAb recognition of tubulin and the neuronal cell surface with initiation of cell signaling and dopamine release supports an emerging theme in autoimmunity whereby cross-reactive or polyreactive autoantibodies against intracellular Ags recognize cell surface epitopes potentially leading to disease.