PHRs: bridging axon guidance, outgrowth and synapse development

https://doi.org/10.1016/j.conb.2009.12.007Get rights and content

Axon guidance, outgrowth, and synapse formation are interrelated developmental events during the maturation of the nervous system. Establishing proper synaptic connectivity requires precise axon navigation and a coordinated switch between axon outgrowth and synaptogenesis. The PHR (human Pam, mouse Phr1, zebrafish Esrom, Drosophila Highwire, and C. elegans RPM-1) protein family regulates both axon and synapse development through their biochemical and functional interactions with multiple signaling pathways. Recent studies have begun to elucidate a common underlying mechanism for PHR functions: Consisting of motifs that affect intracellular signaling, selective protein degradation, and cytoskeleton organization, PHR proteins probably mediate the transition between axon outgrowth and synaptogenesis through integrating intracellular signaling and microtubule remodeling.

Introduction

The establishment of proper synaptic connectivity in the nervous system requires the coordination of intimately coupled developmental processes, axon guidance, outgrowth, and synaptogenesis. Neurons and their projections have the capacity to migrate over long distances in response to guidance cues. Once navigated near their synaptic partners, neurons must transition from a state of outgrowth to that of synaptogenesis, during which a stable connection between the presynaptic and postsynaptic cell forms. Similar transitions are probably recapitulated during the developmental remodeling of the nervous system, as well as repair following nerve injury. In the past decade, the PHR (human Pam, mouse Phr1, zebrafish Esrom, Drosophila Highwire, and C. elegans RPM-1) family proteins have emerged as key regulators for axon guidance, outgrowth, regeneration, and synapse development. Averaging above 4000 amino acids, PHR proteins consist of multiple motifs that suggest their involvement in signaling, scaffolding, as well as ubiquitin-mediated protein degradation. Here we review studies that reveal the complex roles and mechanisms through which PHR proteins regulate axon guidance, synaptogenesis, and regeneration. A common theme emerges that PHR proteins mediate the transition between axon growth and synaptogenesis through promoting the remodeling of microtubule dynamics. We further summarize other functions of PHR proteins, which, in large, remain to be further explored.

Section snippets

A brief history on the discovery of PHRs

The founding member of the PHR family, human Pam (protein associated with Myc), was identified from the Akata Burkitt's lymphoma cell line through its ability to interact with a proto-oncogene Myc [1]. The first insight into the physiological function of PHR proteins came from parallel studies on Caenorhabditis elegans (C. elegans) and Drosophila PHRs, RPM-1 [2••, 3••], and Highwire (HIW) [4••], respectively. Both were identified as regulators of synapse development in genetic screens for

PHR proteins as regulators for synapse development

C. elegans rpm-1 and Drosophila hiw mutants were identified from three independent genetic screens for abnormal axon and/or synapse morphology. In rpm-1 mutants, the PLM mechanosensory neurons often extend their anterior longitudinal processes past synaptic targets, and fail to accumulate vesicles at their axonal branches [3••]. In GABAergic motoneurons however, both overgrown presynaptic terminals, associated with multiple active zones, and axonal regions with underdifferentiated synaptic

PHR proteins as regulators for axon guidance and outgrowth

Although the earlier studies on invertebrate PHR proteins emphasized their roles in synapse formation and growth, defective axonal morphology, notably, the excessive branching of motoneuron axons in hiw mutants [4••], and the aberrant branching, retraction, overgrowth, and target passing in some sensory and motoneuron axons of rpm-1 mutants have been noted [2••, 3••]. These defects were attributed to defective axon termination, which may either contribute to, or be triggered by, a failure in

Invertebrate PHR affects axon guidance, outgrowth and regeneration

Although invertebrate PHR mutants do not exhibit systemic defects in axon guidance and outgrowth, recent studies suggest that RPM-1 activity can modify the phenotype of axon guidance mutants, and vice versa. For example, rpm-1 mutations partially suppress the ventral–dorsal guidance defects in motoneurons when the guidance cue (Netrin/UNC-6) is reduced. Reducing the activity of UNC-5/UNC-5 or Robo/SAX-3 guidance receptors partially suppresses the overextension of mechanosensory axons in rpm-1

PHRs as E3 ubiquitin ligases

The RING-H2 zinc finger motif is a hallmark of E3 ubiquitin ligases. Moreover, many vertebrate and invertebrate PHR mutations affect the RING-H2 domain. hiw mutants exhibit genetic interactions with fat facets, a gene encoding a deubiquitinating enzyme in a dosage-dependent manner, providing the first evidence that HIW functions through ubiquitin-mediated proteasome degradation [20]. The identification of FSN-1 family F-box proteins as the functional partners for PHRs provides further evidence

Convergence on the MAP kinase signaling pathway

What are the targets of this E3 complex during axon or synapse development? In the absence of the ligase activity, the increased level and/or activity of their targets are expected to contribute to defects exhibited by PHR mutants. Genetic and biochemical interactions between PHR mutants and multiple signaling pathways have provided a list of candidates; one conserved target of PHRs is the dual-leucine zipper kinase, DLK-1/Wallenda/DLK, and the respective MAP kinase signaling cascades they

Interactions with other signaling pathways

Consistent with PHR proteins regulating additional targets, PHR mutants exhibit genetic and/or biochemical interactions with components of multiple signaling pathways (Figure 1B). In vivo and in vitro interactions between PHRs and a tumor suppressor protein TSC2/tuberin were noted in several systems. The RING-H2 finger motif of PAM can bind and ubiquitinate TSC2 in vitro [30••]. Phr1 co-immunoprecipitates with the TSC1–TSC2 complex in PC12 cell lines and rat brain, and RNAi-mediated knockdown

A switch between axon guidance, outgrowth and synapse formation

Axon guidance, outgrowth, branching, and synaptogenesis are interconnected developmental events during the maturation of the nervous system. The coordination of these events depends on the spatial regulation of extrinsic cues as well as the temporal regulation of intrinsic programming that dictates an axon's response to cues. PHR mutants exhibit defects in varied combinations of the above events; a likely unifying theme is the failure in transitions between the initiation and termination of

More functions for PHRs?

PHR proteins have additional in vivo functions that are not obviously related to signaling or cytoskeletal dynamics of neurons (Figure 1C). In addition to axon defects, zebrafish esrom mutants exhibit a paler color owing to the decrease of specific pteridines in xanthophores. Esrom is required for the synthesis of tetrahydrobiopterin not only in xanthophores, but also probably in the retinal neurons through unknown mechanisms [38•, 39]. PHR proteins also regulate apoptosis. C. elegans rpm-1 and

Summary and future prospects

PHR proteins have diverse roles in axon guidance, outgrowth, termination, and synaptogenesis. Recent studies have further implicated a role for PHR proteins in axon regeneration. PHR mutants exhibit differences in phenotypes. Nonetheless, PHR proteins may function as regulators to integrate cell signaling and cytoskeletal dynamics through similar mechanisms. It is enticing to speculate that PHRs affect axon and synapse development by promoting cytoskeleton rearrangements in response to

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Anat Kapelnikov and Wesley Hung for comments and suggestion on the review. Michelle Po and Christine Hwang are recipients of the NSERC and OSOTF graduate fellowships, respectively. This work is supported by CIHR grants to Mei Zhen.

References (41)

  • A.M. Schaefer et al.

    rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans

    Neuron

    (2000)
  • J. D'Souza et al.

    Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc)

    Development

    (2005)
  • R.W. Burgess et al.

    Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice

    Mol Cell Biol

    (2004)
  • H. Li et al.

    RPM-1, a Caenorhabditis elegans protein that functions in presynaptic differentiation, negatively regulates axon outgrowth by controlling SAX-3/robo and UNC-5/UNC5 activity

    J Neurosci

    (2008)
  • M. Hammarlund et al.

    Axon regeneration requires a conserved MAP kinase pathway

    Science

    (2009)
  • B. Abrams et al.

    Cellular and molecular determinants targeting the Caenorhabditis elegans PHR protein RPM-1 to perisynaptic regions

    Dev Dyn

    (2008)
  • C. Wu et al.

    Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements

    J Neurosci

    (2005)
  • C. Ehnert et al.

    Protein associated with Myc (PAM) is involved in spinal nociceptive processing

    J Neurochem

    (2004)
  • J.W. Lewcock et al.

    The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics

    Neuron

    (2007)
  • A.J. Bloom et al.

    The requirement for Phr1 in CNS axon tract formation reveals the corticostriatal boundary as a choice point for cortical axons

    Genes Dev

    (2007)
  • Cited by (35)

    • Genetic analysis of synaptogenesis

      2020, Synapse Development and Maturation: Comprehensive Developmental Neuroscience
    • Defining minimal binding regions in Regulator of Presynaptic Morphology 1 (RPM-1) using Caenorhabditis elegans neurons reveals differential signaling complexes

      2017, Journal of Biological Chemistry
      Citation Excerpt :

      These in vivo biochemical results indicate that RPM-1 reduces FSN-1·RAE-1 binding by forming two separate protein complexes with both molecules. C. elegans RPM-1 is a conserved regulator of axon termination and synapse formation (1, 2). Previous work showed that RPM-1 functions as an intracellular signaling hub that positively and negatively regulates numerous downstream signaling pathways (2, 11, 17, 25, 28, 30–32).

    • High myopia-excavated optic disc anomaly associated with a frameshift mutation in the MYC-binding protein 2 gene (MYCBP2)

      2015, American Journal of Ophthalmology
      Citation Excerpt :

      The genetic data therefore suggest that the heterozygous MYCBP2 deletion is the cause of the disease in this family. MYCBP2, or PAM (protein associated with Myc), is a large, highly conserved multifunctional E3 ubiquitin ligase.10 In humans, the highest levels of MYCBP2 mRNA are found in the brain and thymus.26

    • Cadherin-7 regulates mossy fiber connectivity in the cerebellum

      2014, Cell Reports
      Citation Excerpt :

      Previous studies have identified molecules that are involved in the development of both axons and synapses. The conserved PHR family of E3 ubiquitin ligases regulates axon guidance, growth, and synapse formation (Po et al., 2010). The serine/threonine kinase SAD-1 regulates axonal growth termination and presynaptic differentiation in Caenorhabditis elegans (Crump et al., 2001).

    • The Phr1 Ubiquitin Ligase Promotes Injury-Induced Axon Self-Destruction

      2013, Cell Reports
      Citation Excerpt :

      Hence, strategies to manipulate NMNAT2 are particularly attractive therapeutic candidates for treating axonopathies in a broad range of conditions. Phr1 plays an evolutionarily conserved role in various aspects of axon biology (Po et al., 2010). Our findings now demonstrate that PHR proteins play an evolutionarily conserved role in the control of axonal degeneration.

    View all citing articles on Scopus
    View full text