Myostatin: a modulator of skeletal-muscle stem cells.

Myostatin, or GDF-8 (growth and differentiation factor-8), was first identified through sequence identity with members of the BMP (bone morphogenetic protein)/TGF-beta (transforming growth factor-beta) superfamily. The skeletal-muscle-specific expression pattern of myostatin suggested a role in muscle development. Mice with a targeted deletion of the myostatin gene exhibit a hypermuscular phenotype. In addition, inactivating mutations in the myostatin gene have been identified in 'double muscled' cattle breeds, such as the Belgian Blue and Piedmontese, as well as in a hypermuscular child. These findings define myostatin as a negative regulator of skeletal-muscle development. Myostatin binds with high affinity to the receptor serine threonine kinase ActRIIB (activin type IIB receptor), which initiates signalling through a smad2/3-dependent pathway. In an effort to validate myostatin as a therapeutic target in a post-embryonic setting, a neutralizing antibody was developed by screening for inhibition of myostatin binding to ActRIIB. Administration of this antimyostatin antibody to adult mice resulted in a significant increase in both muscle mass and functional strength. Importantly, similar results were obtained in a murine model of muscular dystrophy, the mdx mouse. Unlike the myostatin-deficient animals, which exhibit both muscle hypertrophy and hyperplasia, the antibody-treated mice demonstrate increased musculature through a hypertrophic mechanism. These results validate myostatin inhibition as a therapeutic approach to muscle wasting diseases such as muscular dystrophy, sarcopenic frailty of the elderly and amylotrophic lateral sclerosis.

Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

Cell signalling cascades regulating neuronal growth-promoting and inhibitory cues.

During development of the nervous system, neurons extend axons over considerable distances in a highly stereospecific fashion in order to innervate their targets in an appropriate manner. This involves the recognition, by the axonal growth cone, of guidance cues that determine the pathway taken by the axons. These guidance cues can act to promote and/or repel growth cone advance, and they can act either locally or at a distance from their place of synthesis. The directed growth of axons is partly governed by cell adhesion molecules (CAMs) on the neuronal growth cone that bind to CAMs on the surface of other axons or non-neuronal cells. In vitro assays have established the importance of the CAMs (N-CAM, N-cadherin and the L1 glycoprotein) in promoting axonal growth over cells, such as Schwann cells, astrocytes and muscle cells. Strong evidence now exists implicating the fibroblast growth factor receptor tyrosine kinase as the primary signal transduction molecule in the CAM pathway. Cell adhesion molecules are important constituents of synapses, and CAMs appear to play important and diverse roles in regulating synaptic plasticity associated with learning and memory. Negative extracellular signals which physically direct neurite growth have also been described. The latter include the neuronal growth inhibitory proteins Nogo and myelin-associated glycoprotein, as well as the growth cone collapsing Semaphorins/neuropilins. Although less well characterised, evidence is now beginning to emerge describing a role for Rho kinase-mediated signalling in inhibition of neurite outgrowth. This review focuses on some of the major themes and ideas associated with this fast-moving field of neuroscience.

Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth.

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition.

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.

Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.

Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

Novel drug development for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has become an increasingly attractive area for the pharmaceutical industry, the most experimentally tractable of the neurodegenerative diseases. Mechanisms underlying cell death in ALS are likely to be important in more common but more complex disorders. Riluzole, the only drug launched for treatment ALS is currently undergoing industrial trials for Alzheimer's, Parkinson's, Huntington disease, stroke and head injury. Other compounds in Phase III testing for ALS (mecamserin, xaliproden, gabapentin) are also in trials for other neurodegenerative disorders. Mechanisms of action of these advanced compounds are limited to glutamate antagonism, direct or indirect growth factor activity, as well as GABA agonism and interaction with calcium channels. A broader range of mechanisms is represented by compounds in Phase I trials: glutamate antagonism (dextramethorphan/p450 inhibitor; talampanel), growth factors (leukemia inhibiting factor; IL-1 receptor; encapsulated cells secreting CNTF) and antioxidants (TR500, a glutathione-repleting agent; recombinant superoxide dismutase; procysteine.) An even broader range of mechanisms is being explored in preclinical discovery programs. Recognition of the difficulties associated with delivery of protein therapeutics to the CNS has led to development of small molecules interacting either with neurotrophin receptors or with downstream intracellular signalling pathways. Other novel drug targets include caspaces, protein kinases and other molecules influencing apoptosis. High-throughput screens of large libraries of small molecules yield lead compounds that are subsequently optimized by chemists, screened for toxicity, and validated before a candidate is selected for clinical trials. The net is cast wide in early discovery efforts, only about 1% of which result in useful drugs at the end of a decade-long process. Successful discovery and development of novel drugs will increasingly depend on collaborative efforts between the academy and industry.

ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site.

Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.

The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects.

Basic fibroblast growth factor (FGF-2) promotes survival and/or neurite outgrowth from a variety of neurons in cell culture and regenerative processes in vivo. FGFs exert their effects by activating cell surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not been characterized on neuronal cell behaviors to date. In the present study, we show that the FGFR1 inhibitor PD 173074 potently and selectively antagonized the neurotrophic and neurotropic actions of FGF-2. Nanomolar concentrations of PD 173074 prevented FGF-2, but not insulin-like growth factor-1, support of cerebellar granule neuron survival under conditions of serum/K(+) deprivation; another FGF-2 inhibitor, SU 5402, was effective only at a 1,000-fold greater concentration. Neither PD 173074 nor SU 5402, at 100 times their IC(50) values, interfered with the survival of dorsal root ganglion neurons promoted by nerve growth factor, ciliary neurotrophic factor, or glial cell line-derived neurotrophic factor. PD 173074 and SU 5402 displayed 1,000-fold differential IC(50) values for inhibition of FGF-2-stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF-2-induced mitogen-activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF-2. PD 173074 represents a valuable tool for dissecting the role of FGF-2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.

Cellular uptake and spread of the cell-permeable peptide penetratin in adult rat brain.

Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell-permeable peptide consisting of a 16-amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion-fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 microg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose-dependent fashion. Doses of 1 microg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 microg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.

Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.

Ectopic expression of NCAM in skeletal muscle of transgenic mice results in terminal sprouting at the neuromuscular junction and altered structure but not function.

The neuromuscular system provides an excellent model for the analysis of molecular interactions involved in the development and plasticity of synaptic contacts. The neural cell adhesion molecule (NCAM) is believed to be involved in the development and plasticity of the neuromuscular junction, in particular the axonal sprouting response observed in paralyzed and denervated muscle. In order to explore the role of myofiber NCAM in modulating the differentiation of motor neurons, we generated transgenic mice expressing a GPI-anchored NCAM isoform that is normally found in developing and denervated muscle, under the control of a skeletal muscle-specific promoter. This results in the constitutive expression of NCAM at postnatal ages, a time when the endogenous mouse NCAM is absent from the myofiber. We found that a significant number of neuromuscular junctions in adult transgenic animals displayed terminal sprouting (>20%) reminiscent of that elicited in response to cessation of neuromuscular activity. Additionally, a significant increase in the size and complexity of neuromuscular synapses as a result of extensive intraterminal sprouting was detected. Electrophysiological studies, however, revealed no significant alterations of neuromuscular transmission at this highly efficient synapse. Sprouting in response to paralysis or following nerve crush was also significantly enhanced in transgenic animals. These results suggest that in this ectopic expression model NCAM can directly modulate synaptic structure and motor neuron-muscle interactions. The results contrast with knockout experiments of the NCAM gene, where very limited changes in the neuromuscular system were observed.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Differential display of genes expressed at the midbrain - hindbrain junction identifies sprouty2: an FGF8-inducible member of a family of intracellular FGF antagonists.

Specification and polarization of the midbrain and anterior hindbrain involve planar signals originating from the isthmus. Current evidence suggests that FGF8, expressed at the isthmus, provides this patterning influence. In this study, we have sought to identify novel genes which are involved in the process by which regional identity is imparted to midbrain and anterior hindbrain (rhombomere 1). An enhanced differential display reverse transcription method was used to clone cDNAs derived from transcripts expressed specifically in either rhombomere 1 or midbrain during the period of isthmic patterning activity. This gene expression screen identified 28 differentially expressed cDNAs. A clone upregulated in cDNA derived from rhombomere 1 tissue showed a 91% identity at the nucleotide level to the putative human receptor tyrosine kinase antagonist: sprouty2. In situ hybridization on whole chick embryos showed chick sprouty2 to be expressed initially within the isthmus and rhombomere 1, spatially and temporally coincident with Fgf8 expression. However, at later stages this domain was more extensive than that of Fgf8. Introduction of ligand-coated beads into either midbrain or hindbrain region revealed that sprouty2 could be rapidly induced by FGF8. These data suggest that sprouty2 participates in a negative feedback regulatory loop to modulate the patterning activity of FGF8 at the isthmus.

Heteromeric assembly of GABA(B)R1 and GABA(B)R2 receptor subunits inhibits Ca(2+) current in sympathetic neurons.

Neuronal GABA(B) receptors regulate calcium and potassium currents via G-protein-coupled mechanisms and play a critical role in long-term inhibition of synaptic transmission in the CNS. Recent studies have demonstrated that assembly of GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits into functional heterodimers is required for coupling to potassium channels in heterologous systems. However whether heterodimerization is required for the coupling of GABA(B) receptors to effector systems in neurons remains to be established. To address this issue, we have studied the coupling of recombinant GABA(B) receptors to endogenous Ca(2+) channels in superior cervical ganglion (SCG) neurons using nuclear microinjection to introduce both sense and antisense expression constructs. Patch-clamp recording from neurons injected with both GABA(B)R1a/1b and GABA(B)R2 cDNAs or with GABA(B)R2 alone produced marked baclofen-mediated inhibition of Ca(2+) channel currents via a pertussis toxin-sensitive mechanism. The actions of baclofen were blocked by CGP62349, a specific GABA(B) antagonist, and were voltage dependent. Interestingly, SCGs were found to express abundantly GABA(B)R1 but not GABA(B)R2 at the protein level. To determine whether heterodimerization of GABA(B)R1 and GABA(B)R2 subunits was required for Ca(2+) inhibition, the GABA(B)R2 expression construct was microinjected with a GABA(B)R1 antisense construct. This resulted in a dramatic decrease in the levels of the endogenous GABA(B)R1 protein and a marked reduction in the inhibitory effects of baclofen on Ca(2+) currents. Therefore our results suggest that in neurons heteromeric assemblies of GABA(B)R1 and GABA(B)R2 are essential to mediate GABAergic inhibition of Ca(2+) channel currents.

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32.

BACKGROUND: Neurons project their axons along specific pathways in order to establish appropriate connections with their target cells. The rate and direction of axonal growth is determined by interactions between the highly motile growth cone and environmental cues that can act in either an attractive or a repulsive manner. Locomotion is ultimately dependent upon the reorganisation of the actin cytoskeleton and an established role for the Rho family of small GTPases in regulating this process in non-neuronal cells identifies them as candidate signalling molecules in growth cones. An inactive form of Rac1 has recently been shown to inhibit the 'growth-cone collapse' response induced by chick Sema3A, a protein that has recently been established as an important guidance cue. The molecular basis for this inhibition remains unclear. RESULTS: We have made a series of overlapping peptides from the amino-terminal region of Rac1 and rendered them cell permeable by synthesis in tandem with an established internalisation vector. We report here that a peptide encompassing Rac1 amino acids 17-32 binds directly to the established Rac1-interacting molecules PAK, WASP, 3BP-1 and p85beta(P13K), but not to p67(Phox). Furthermore, the peptide can compete with activated Rac1 for target binding, and inhibits Sema3A-induced growth-cone collapse. We also synthesised cell-permeable peptides that correspond to the Cdc42/Rac1-binding (CRIB) motifs present in PAK and N-WASP. Our results show that a CRIB-containing peptide from PAK, but not that from N-WASP, inhibits growth-cone collapse and that the inhibitory activity correlates with binding to Rac1 and not to Cdc42. CONCLUSIONS: Our results suggest that Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32, and demonstrate the feasibility of designing new cell-permeable inhibitors of small GTPases.