Analgesic efficacy and safety of the novel p38 MAP kinase inhibitor, losmapimod, in patients with neuropathic pain following peripheral nerve injury: a double-blind, placebo-controlled study.

BACKGROUND: Inhibitors of p38 mitogen-activated protein kinase are undergoing evaluation as a novel class of anti-rheumatic drugs, by virtue of their ability to suppress the production of pro-inflammatory cytokines. Emerging data suggests that they may also attenuate peripheral or central sensitization in neuropathic pain. A double-blind, placebo-controlled study was undertaken to evaluate the analgesic efficacy of losmapimod (GW856553), a novel p38α/ß inhibitor, in subjects with neuropathic pain following traumatic peripheral nerve injury. METHODS: One hundred and sixty-eight subjects with pain of at least moderate intensity (average daily score ≥4 on an 11-point pain intensity numeric rating scale; PI-NRS) at baseline were randomized to receive oral losmapimod, 7.5 mg BID or placebo for 28 days. Efficacy and safety assessments were undertaken at weekly clinic visits. RESULTS: The mean treatment difference for the change in average daily pain score from baseline to week 4 of treatment based on the PI-NRS was -0.22 (95% CI -0.73, 0.28) in favour of losmapimod over placebo (p = 0.39). There were no statistically significant or clinically meaningful differences between the treatment groups over the 4-week dosing period for either the primary or secondary efficacy variables. There were no unexpected safety or tolerability findings following dosing with losmapimod. CONCLUSIONS: Losmapimod could not be differentiated from placebo in terms of a primary analgesia response in patients with pain following peripheral nerve injury. The lack of response could reflect inadequate exposure at central sites of action or differences between rodent and human with respect to the target or neuropathic pain mechanisms.

Evolution of the spectrin-based membrane skeleton.

A group of four proteins - spectrin, ankyrin, 4.1 and adducin - evolved with the metazoa. These membrane-cytoskeletal proteins cross-link actin on the cytoplasmic face of plasma membranes and link a variety of transmembrane proteins to the cytoskeleton. In this paper, the evolution of these proteins is analysed. Genomics indicate that spectrin was the first to appear, since the genome of the choanoflagellate Monosiga brevicolis contains genes for alpha, beta and betaH spectrin. This organism represents a lineage of free-living and colonial protists from which the metazoa are considered to have diverged. This indicates that spectrin emerged in evolution before the animals. Simple animals such as the placozoan Trichoplax adherens also contain recognizable precursors of 4.1, ankyrin and adducin, but these could probably not bind spectrin. Ankyrin and adducin seem to have acquired spectrin-binding activity with the appearance of tissues since they appear to have largely the same domain structure in all eumetazoa. 4.1 was adapted more recently, with the emergence of the vertebrates, to bind spectrin and promote its interaction with actin. A simple hypothesis is that spectrin was prerequisite (but not sufficient) for animal life; that spectrin interaction with ankyrin and adducin was required for evolution of major tissues; and that 4.1 acquired a spectrin-actin binding activity as animal size increased with the appearance of vertebrates. The spectrin/ankyrin/adducin/4.1 complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Characterization of the cell membrane-associated products of the Neuregulin 4 gene.

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment.

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical (32)P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.

The postsynaptic spectrin/4.1 membrane protein "accumulation machine".

An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that beta spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, alpha-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.

Properties of the C-terminal domain of 4.1 proteins.

At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.

Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues.

The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

Protein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteins.

4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80-kDa 4.1R polypeptide is enriched 11-fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of approximately 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti-4.1G antibodies; the 200 kDa species is enriched 2.5-fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R-interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and alpha-internexin, respectively. A complex containing 80 kDa 4.1R, alpha-internexin and neurofilament L was immunoprecipitated with anti-4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities.

Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activities.

It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Ca2+-dependent interaction with calmodulin is conserved in the synapsin family: identification of a high-affinity site.

The synapsins are a family of proteins associated with small synaptic vesicles that are implicated in synaptic maintenance and in the supply of vesicles for exocytosis. They are well characterized as substrates for protein kinases, and one class of synapsin, synapsin I, has been shown to bind, and be regulated by, calmodulin. A representative of the synapsin II class is now shown to bind calmodulin. Optical biosensor assays of Ca2+-dependent calmodulin binding to recombinant rat synapsin IIb indicated an apparent KD for calmodulin of 31 +/- 5 nM. Phosphorylation at Ser 10 increased the rates of calmodulin association (by a factor of 10) and dissociation (by a factor of 20). Fragment analysis and predictions from the sequence indicated two potential calmodulin binding sequences in the conserved central (C) domain. Peptides representing these sequences (residues 122-143 and 313-334 in synapsin IIb) were synthesized. Peptide 122-143 was found to bind calmodulin (KD 32 +/- 10 nM) and inhibit interaction of synapsin IIb with calmodulin. The interaction of peptide 313-334 was much weaker. Sequences similar to residues 122-143 are present in all published synapsin sequences. Calmodulin binding by synapsins seems not to be confined to mammals: a recombinant Drosophila synapsin 1 fragment containing part of the C-domain showed Ca2+-dependent binding to mammalian calmodulin. We conclude that calmodulin binding to synapsins is likely to be a general aspect of regulation of synaptic function.

Definition of a sequence unique in beta II spectrin required for its axon-specific interaction with fodaxin (A60).

Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon-specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of alpha I, beta I, and beta II polypeptides) but not erythrocyte spectrin (alpha I and beta I). Because alpha II and beta II spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain alpha chains. Bacterially expressed C-terminal segments 18-19 of beta II spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18-19 of the somatodendritic beta I sigma 2 spectrin showed no interaction with fodaxin. Within beta II, fodaxin binding activity was localized to residues 2,087-2,198, which are unique to beta II and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of beta II and beta I sigma 2 are candidates to mediate the isotype-specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of beta spectrin isoforms.

Phenotypic morphology and the expression of cytoskeletal markers during long-term differentiation of human SH-SY5Y neuroblastoma cells.

A simple method for differentiating human-derived SH-SY5Y neuroblastoma cells to provide a stable, mature, neuronal morphology is described. SH-SY5Y cells can be induced to differentiate terminally with retinoic acid in medium with low levels of serum. The morphological differentiation of the parental cell line SK-N-SH was compared with that seen in the SH-SY5Y cells. Changes in the cytoskeleton of SH-SY5Y cells indicated that differentiation proceeds continuously over the 1-month period studied after initiating differentiation. Immunoblot analysis demonstrated increased expression of the high molecular weight neurofilament polypeptide NF-H, the microtubule-associated protein tau, and the synaptic vesicle-associated protein synapsin I, indicating that an increasingly mature, neuronal phenotype was being expressed. The cultures were not dependent on retinoic acid for continued survival. SH-SY5Y cultures differentiated over extended periods should provide a good in vitro model for studying the neurotoxic potential of compounds and mechanisms of toxicity, particularly in longer-term or multiple exposure studies, for example on cytoskeletal function, where acute toxicity is not the aspect of interest.