Presynaptic terminals contain highly organized subcellular structures to facilitate neurotransmitter release. In C. elegans, the typical presynaptic terminal has an electron-dense active zone surrounded by synaptic vesicles. Loss-of-function mutations in the rpm-1 gene result in abnormally structured presynaptic terminals in GABAergic neuromuscular junctions (NMJs), most often manifested as a single presynaptic terminal containing multiple active zones. The RPM-1 protein has an RCC1-like guanine nucleotide exchange factor (GEF) domain and a RING-H2 finger. RPM-1 is most similar to the Drosophila presynaptic protein Highwire (HIW) and the mammalian Myc binding protein Pam. RPM-1 is localized to the presynaptic region independent of synaptic vesicles and it functions cell autonomously. The temperature-sensitive period of rpm-1 coincides with the time of synaptogenesis. rpm-1 may regulate the spatial arrangement, or restrict the formation, of presynaptic structures (Zhen, 2000).
rpm-1 mutations were isolated based on the abnormal morphologies of presynaptic terminals visualized by the synaptic vesicle-tagged GFP markers expressed in different types of neurons, hence the name regulator of presynaptic morphology. This analysis of the GABAergic NMJs has revealed that loss of rpm-1 function causes two types of defects: overdeveloped presynaptic terminals that contain multiple presynaptic active zones and underdeveloped presynaptic terminals that have few synaptic vesicles. In the strongest mutant background, both types of abnormality are present, and the overdeveloped presynaptic terminals appear to be predominant (Zhen, 2000).
rpm-1 is widely expressed in the nervous system. Different types of neurons appear to be affected in different manners and to different extents in rpm-1 mutants. The defects in cholinergic NMJs are similar to, but less severe than, those of GABAergic NMJs, and the abnormal presynaptic terminals either contain few vesicles or have elongated presynaptic active zones. No obvious axonal morphological defects are observed in GABAergic and cholinergic motor neurons of the ventral nerve cord. By contrast, the axons of mechanosensory and SAB motor neurons have extra branches, often bypass their targets, and make few synapses. It is not known whether the synapses of these mechanosensory and SAB neurons are affected at the ultrastructural level. Such 'bypass' phenotypes could be caused by a failure in recognizing targets or could be a secondary effect of failures in the initiation and stabilization of synapse formation. Formation of NMJs between ventral cord motor neurons and body muscles does not depend critically on the correct axon pathfinding of motor neurons and appears to be initiated by motor neurons responding to signals from muscle arms. However, mechanosensory and SAB neurons may form synapses onto their targets only after they are guided to the target regions. The different mutant phenotypes in mechanosensory and motor neurons may reflect the differences in how synapses are formed in different neurons or may indicate that rpm-1 has different functions in different neurons (Zhen, 2000).
Little is known of mechanisms regulating presynaptic differentiation. rpm-1 was identified in a screen for mutants with defects in patterning of a presynaptic marker at certain interneuronal synapses. The predicted RPM-1 protein contains zinc binding, RCC1, and other conserved motifs. In rpm-1 mutants, mechanosensory neurons fail to accumulate tagged vesicles, retract synaptic branches, and ectopically extend axons. Some motor neurons branch and overgrow; others show altered synaptic organization. Expression of RPM-1 in the presynaptic mechanosensory neurons is sufficient to rescue phenotypes in these cells. Certain rpm-1 phenotypes are temperature sensitive, revealing that RPM-1 function can be bypassed by maintaining mutants at the permissive temperature at stages commensurate with synapse formation in wild-type animals. These results indicate that RPM-1 functions cell autonomously during synaptogenesis to regulate neuronal morphology (Schaefer, 2000).
What mechanism in the presynaptic mechanosensory neurons is regulated in vivo by RPM-1? There are at least two possibilities. First, RPM-1 could regulate neuronal maturation. The data are consistent with a model whereby mechanosensory synapses fail to mature, generating retraction of the synaptic branch. Although fewer synaptic branches are observed than in wild type, additional transient branches could be missed in the analyses of staged populations. Such growth cone dynamics would be consistent with the 20-55 µm/hr pace of VD motor neuron migration. Moreover, if RPM-1 is a regulator of synaptic maturation, the rpm-1 mutant phenotype of ectopic axonal targeting could be secondary to failure to form stable synapses. Perhaps such failure generates a retrograde signal, which leads to ectopic growth toward the VNC. Alternatively, the ectopic growth could be a primary defect, resulting from failure of the axonal growth cone to mature into a stable and static structure in the lateral midbody region (Schaefer, 2000).
In a different scenario, RPM-1 could be involved in mechanisms of outgrowth at the appropriate subcellular locus. Normally, PLM extends a synaptic branch perpendicularly from the middle of the axon, in a defined region between PVM and the vulva. Perhaps, in rpm-1 mutants, the machinery for synaptic branch extension is misrouted to the inappropriate intracellular location. However, such machinery could normally be distributed uniformly along the axon, but local cues regulating the locus of branch extension are somehow disrupted in rpm-1 animals. Normal and ectopic targeting are not mutually exclusive: a single PLM neuron can display both a synaptic branch and ectopic growth from the end of the axon (Schaefer, 2000).
The first hypothesis, that RPM-1 is part of a mechanism to effect neuronal maturation, is favored. This explanation is more consistent with the phenotype of synaptic branch retraction. Moreover, a role in maturation is compatible with the motor neuron phenotypes. The SAB motor neurons in rpm-1 sprout additional branches, similar to sprouting in SAB neurons induced by deficits in synaptic activity. In the DNC neuropil, there were fewer presynaptic densities. Wider gaps between these labeled presynaptic specializations could simply reflect that many specializations are missing in mutant animals. Labeled presynaptic specializations in the DNC appear to aggregate in mutants. Perhaps these aggregations represent the addition of active zones within individual motor neurons. Alternatively, they may be generated by a redistribution of existing synapses (Schaefer, 2000).
Observations of these changes in presynaptic labeling and in morphology of mechanosensory and motor neurons thus may reflect a primary defect in synapse formation. Failure of synapses to form or to mature may, in turn, generate distinct cellular responses, depending on cell type (Schaefer, 2000).
If indeed RPM-1 is involved in neuronal maturation at the time of synaptogenesis, its function is not absolutely required: labeling with synaptic markers indicates that many synapses do differentiate in rpm-1 animals. Neuronal patterning is grossly normal. For the screen, a gross behavioral phenotype from loss, per se, of chemical synapses made by mechanosensory neurons was not predicted; these synapses are not required for the touch response. Nevertheless, the essentially normal behavior of mutant animals confirms that there are no general synaptic defects (Schaefer, 2000).
During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-beta family members between presynaptic and postsynaptic termini are proposed to govern synapse formation. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. FSN-1 is a novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. Evidence is provided that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation (Liao, 2004).
Genetic studies using a set of overlapping deletions centered at the piebald locus on distal mouse chromosome 14 have defined a genomic region associated with respiratory distress and lethality at birth. The candidate gene Phr1 that is located within the respiratory distress critical genomic interval has been isolated and characterized. Phr1 is the ortholog of the human Protein Associated with Myc as well as Drosophila highwire and Caenorhabditis elegans regulator of presynaptic morphology 1. Phr1 is expressed in the embryonic and postnatal nervous system. In mice lacking Phr1, the phrenic nerve fails to completely innervate the diaphragm. In addition, nerve terminal morphology is severely disrupted, comparable to the synaptic defects seen in the Drosophila hiw and C. elegans rpm-1 mutants. Although intercostal muscles were completely innervated, they also showed dysmorphic nerve terminals. In addition, sensory neuron terminals in the diaphragm were abnormal. The neuromuscular junctions showed excessive sprouting of nerve terminals, consistent with inadequate presynaptic stimulation of the muscle. On the basis of the abnormal neuronal morphology seen in mice, Drosophila, and C. elegans, it is proposed that Phr1 plays a conserved role in synaptic development and is a candidate gene for respiratory distress and ventilatory disorders that arise from defective neuronal control of breathing (Burgess, 2004).
Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, it was established that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones that is required for topographic map formation (D'Souza, 2005).
Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. The pigment phenotype was investigated and, from this, evidence is provided for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis (Le Guyader, 2005).
Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1lf mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. These data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade (Nakata, 2005).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.