Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth

N-cadherin is the predominant mediator of calcium-dependent adhesion in the nervous system (Takeichi, M. 1988. Development (Camb.). 102: 639-655). Investigations using antibodies to block N-cadherin function (Bixby, J.L., R.L. Pratt, J. Lilien, and L.F. Reichardt. 1987. Proc. Natl. Acad. Sci. USA. 84:2555-2569; Bixby, J.L., J. Lilien, and L.F. Reichardt. 1988. J. Cell Biol. 107:353-362; Tomaselli, K.J., K.N. Neugebauer, J.L. Bixby, J. Lilien, and L.F. Reichardt. 1988. Neuron. 1:33-43) or transfection of the N-cadherin gene into heterologous cell lines (Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi. 1988. Nature (Lond.). 334:62-64) have provided evidence that N-cadherin, alone or in combination with other molecules, can participate in the induction of neurite extension. We have developed an affinity purification procedure for the isolation of whole N-cadherin from chick brain and have used the isolated protein as a substrate for neurite outgrowth. N-cadherin promotes the rapid extension of neurites from chick ciliary ganglion neurons, which extend few or no neurites on adhesive but noninducing substrates such as polylysine, tissue culture plastic, and collagens. N-cadherin is extremely potent, more so than the L1 adhesion molecule, and comparable to the extracellular matrix protein laminin. Compared to laminin, however. N-cadherin promotes outgrowth from ciliary ganglion neurons extremely rapidly and with a distinct morphology. These results provide a direct demonstration that N-cadherin is sufficient to induce neurite outgrowth when substrate bound and suggest that the mechanism(s) involved may differ from that induced by laminin.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture

BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.     

Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.

A random-primed cDNA expression library constructed from the mRNA of neuroblastoma cells (NG108) was used to clone a specific rabies virus (RV) receptor. A soluble form of the RV glycoprotein (Gs) was utilized as a ligand to detect positive cells. We identified the murine low-affinity nerve-growth factor receptor, p75NTR. BSR cells stably expressing p75NTR were able to bind Gs and G-expressing lepidopteran cells. The ability of the RV glycoprotein to bind p75NTR was dependent on the presence of a lysine and arginine in positions 330 and 333 respectively of antigenic site III, which is known to control virus penetration into motor and sensory neurons of adult mice. P75NTR-expressing BSR cells were permissive for a non-adapted fox RV isolate (street virus) and nerve growth factor (NGF) decreased this infection. In infected cells, p75NTR associates with the RV glycoprotein and could be precipitated with anti-G monoclonal antibodies. Therefore, p75NTR is a receptor for street RV.     

Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors.

Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Identification of twinfilin-2 as a factor involved in neurite outgrowth by RNAi-based screen

Using RNA interference (RNAi) to suppress gene expression, we attempted to identify tyrosine kinases involved in the extension of neurites from SH-SY5Y cells. A comprehensive analysis of gene “knock-down” profiles with small interfering RNAs (siRNAs) revealed candidate proteins that might control neurite extension. Phenotype-based screening of differentiating SH-SY5Y cells following retinoic acid (RA) stimulation indicated that twinfilin-2 is a protein that is involved in neurite outgrowth, as confirmed by morphological analysis of twinfilin-2-overexpressing cells.     

Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival

Recently, mutations in the progranulin (PGRN) gene were found to cause familial and apparently sporadic frontotemporal lobe dementia (FTLD). Moreover, missense changes in PGRN were identified in patients with motor neuron degeneration, a condition that is related to FTLD. Most mutations identified in patients with FTLD until now have been null mutations. However, it remains unknown whether PGRN protein levels are reduced in the central nervous system from such patients. The effects of PGRN on neurons also remain to be established. We report that PGRN levels are reduced in the cerebrospinal fluid from FTLD patients carrying a PGRN mutation. We observe that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons. These results demonstrate that PGRN/GRN is a neurotrophic factor with activities that may be involved in the development of the nervous system and in neurodegeneration.     

A simple, flexible, nonfluorescent system for the automated screening of neurite outgrowth

Measurement of neurite outgrowth is a common assay of neurotrophic activity. However, currently available techniques for measuring neurite outgrowth are either time or resource intensive. The authors established a system in which chronic treatment of a subcloned SH-SY5Y cell line with aphidicolin and various concentrations of nerve growth factor (NGF) induced discernable alterations in proliferation and differentiation. Cells were fixed, labeled with a nonfluorescent dye, and evaluated both manually and with an automated analysis system. NGF increased multiple parameters of differentiation, including neurite length, the proportion of cells extending neurites, and branching, as well as promoting cellular survival/proliferation. Interestingly, although NGF treatment increased the total number of branches, it actually decreased the proportion of branches per neurite length. The authors observed no differences in results obtained using the manual and automated systems, but the automated system was orders of magnitude faster. To demonstrate the flexibility of the system, the authors also show that they could measure changes in differentiation induced by a small-molecule Rho kinase inhibitor, as well as by retinoic acid cotreatment with brain-derived neurotrophic factor. In addition to this flexibility, this system does not require specialized equipment or fluorescent antibodies for analysis and therefore provides a less resource-intensive alternative to fluorescence-based systems.     

Central nervous system mechanisms for pain modulation

Although a great deal has been learned about the neural basis for stimulation-produced analgesia, it is evident that the 'analgesia systems' are much more complex than was initially thought. Part of the complexity derives from the fact that a number of different pathways, using several different neurotransmitters, can affect nociceptive transmission. Further complexity stems from evidence that nociceptive transmission can be modulated both at a spinal cord level and at higher levels of the nociceptive projection system, such as the thalamus. Hopefully, a greater understanding of the 'analgesia systems' will lead to explanations for a number of puzzling aspects of pain and perhaps to improved therapy.     

Ependymin as a substrate for outgrowth of axons from cultured explants of goldfish retina

Ependymin, a prominent protein of the brain's extracellular fluid (ECF) was previously implicated in the consolidation of memory and in the activity-driven sharpening of the retinotectal projection. Because both these phenomena probably involve the growth and elaboration of appropriate synapses, we have tested whether ependymin can serve as a substrate for the growth of axons from goldfish retinal ganglion cells in a culture assay. Ependymin (Ep), laminin (LAM), polylysine (PL), and Concanavalin A (Con A) were plated on glass coverslips either uniformly or in striped patterns. Ep alone, either soluble or partly polymerized (by dropping calcium concentration and pH), was a good substrate for axonal outgrowth, as good or better than PL and Con A, but not as good as LAM. Neurites grew faster on LAM (71 microns/h) than on Ep (32 microns/h) or on PL (22 microns/h). Fasciculation was low on LAM, intermediate on Ep, and highest on PL. In exclusive side-by-side stripe assays, axons preferred LAM over Ep, but gave weak or no preference for Ep over Con A or PL. With stripes of LAM + Ep alongside pure LAM, the axons preferred the mixture of LAM + Ep. When antibodies to Ep were plated in stripes over continuous Ep substrate, the axons avoided the antibody-blocked stripes and grew on the Ep stripes. Antibodies to Ep did not, however, block growth on laminin substrates, nor did antibodies to LAM block growth on Ep. Dot blots and western blots showed very little cross recognition between the antibodies. Ependymin is a good substrate for neurite outgrowth, which is normally present in ECF, and adhesion to Ep is independent of LAM and possibly additive to it.     

Expression and purification of recombinant tristetraprolin that can bind to tumor necrosis factor-alpha mRNA and serve as a substrate for mitogen-activated protein kinases

Tristetraprolin (TTP) is an mRNA-binding protein, but studies of this interaction have been difficult due to problems with the purification of recombinant TTP. In the present study, we expressed human and mouse TTP as glutathione S-transferase and maltose-binding protein (MBP) fusion proteins in Escherichia coli, and purified them by affinity resins and Mono Q chromatography. TTP cleaved from the fusion protein was identified by immunoblotting, MALDI-MS, and protein sequencing, and was further purified to homogeneity by continuous-elution SDS-gel electrophoresis. Purified recombinant TTP bound to the AU-rich element of tumor necrosis factor-alpha (TNFalpha) mRNA and this binding was dependent on Zn(2+). Results from sizing columns suggested that the active species might be in the form of an oligomer of MBP-TTP. Recombinant TTP was phosphorylated by three members of the mitogen-activated protein (MAP) kinase family, p42, p38, and JNK, with half-maximal phosphorylation occurring at approximately 0.5, 0.25, and 0.25 microM protein, respectively. Phosphorylation by these kinases did not appear to affect the ability of TTP to bind to TNFalpha mRNA under the assay conditions. This study describes a procedure for purifying nonfusion protein TTP to homogeneity, demonstrates that TTP's RNA-binding activity is zinc dependent, and that TTP can be phosphorylated by JNK as well as by the other members of the greater MAP kinase family.     

Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Here, we report that mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling.