Philip F. Copenhaver

Amyloid Precursor Proteins Interact with the Heterotrimeric G Protein Go in the Control of Neuronal Migration

Amyloid precursor protein (APP) belongs to a family of evolutionarily conserved transmembrane glycoproteins that has been proposed to regulate multiple aspects of cell motility in the nervous system. Although APP is best known as the source of β-amyloid fragments (Aβ) that accumulate in Alzheimers disease, perturbations affecting normal APP signaling events may also contribute to disease progression. Previous in vitro studies showed that interactions between APP and the heterotrimeric G protein Goα-regulated Goα activity and Go-dependent apoptotic responses, independent of Aβ. However, evidence for authentic APP–Go interactions within the healthy nervous system has been lacking. To address this issue, we have used a combination of in vitro and in vivo strategies to show that endogenously expressed APP family proteins colocalize with Goα in both insect and mammalian nervous systems, including human brain. Using biochemical, pharmacological, and Bimolecular Fluorescence Complementation assays, we have shown that insect APP (APPL) directly interacts with Goα in cell culture and at synaptic terminals within the insect brain, and that this interaction is regulated by Goα activity. We have also adapted a well characterized assay of neuronal migration in the hawkmoth Manduca to show that perturbations affecting APPL and Goα signaling induce the same unique pattern of ectopic, inappropriate growth and migration, analogous to defective migration patterns seen in mice lacking all APP family proteins. These results support the model that APP and its orthologs regulate conserved aspects of neuronal migration and outgrowth in the nervous system by functioning as unconventional Goα-coupled receptors.

How To Innervate A Simple Gut: Familiar Themes And Unique Aspects In The Formation Of The Insect Enteric Nervous System

Like the vertebrate enteric nervous system (ENS), the insect ENS consists of interconnected ganglia and nerve plexuses that control gut motility. However, the insect ENS lies superficially on the gut musculature, and its component cells can be individually imaged and manipulated within cultured embryos. Enteric neurons and glial precursors arise via epithelial‐to‐mesenchymal transitions that resemble the generation of neural crest cells and sensory placodes in vertebrates; most cells then migrate extensive distances before differentiating. A balance of proneural and neurogenic genes regulates the morphogenetic programs that produce distinct structures within the insect ENS. In vivo studies have also begun to decipher the mechanisms by which enteric neurons integrate multiple guidance cues to select their pathways. Despite important differences between the ENS of vertebrates and invertebrates, common features in their programs of neurogenesis, migration, and differentiation suggest that these relatively simple preparations may provide insights into similar developmental processes in more complex systems. Deveopmental Dynamics 236:1841–1864, 2007.

A translational continuum of model systems for evaluating treatment strategies in Alzheimer’s disease: isradipine as a candidate drug

SUMMARY A growing body of evidence supports the ‘calcium hypothesis’ of Alzheimer’s disease (AD), which postulates that a variety of insults might disrupt the homeostatic regulation of neuronal calcium (Ca2+) in the brain, resulting in the progressive symptoms that typify the disease. However, despite ongoing efforts to develop new methods for testing therapeutic compounds that might be beneficial in AD, no single bioassay permits both rapid screening and in vivo validation of candidate drugs that target specific components of the Ca2+ regulatory machinery. To address this issue, we have integrated four distinct model systems that provide complementary information about a trial compound: the human neuroblastoma MC65 line, which provides an in vitro model of amyloid toxicity; a transgenic Drosophila model, which develops age-dependent pathologies associated with AD; the 3×TgAD transgenic mouse, which recapitulates many of the neuropathological features that typify AD; and the embryonic nervous system of Manduca, which provides a novel in vivo assay for the acute effects of amyloid peptides on neuronal motility. To demonstrate the value of this ‘translational suite’ of bioassays, we focused on a set of clinically approved dihydropyridines (DHPs), a class of well-defined inhibitors of L-type calcium channels that have been suggested to be neuroprotective in AD. Among the DHPs tested in this study, we found that isradipine reduced the neurotoxic consequences of β-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca2+ hypothesis of AD, and indicate that isradipine represents a promising drug for translation into clinical trials. In addition, these studies also demonstrate that this continuum of bioassays (representing different levels of complexity) provides an effective means of evaluating other candidate compounds that target specific components of the Ca2+ regulatory machinery and that therefore might be beneficial in the treatment of AD.

Remodeling of motor terminals during metamorphosis of the moth Manduca sexta: expression patterns of two distinct isoforms of Manduca fasciclin II.

During metamorphosis of the moth Manduca sexta, the neuromuscular system of the thoracic legs is reorganized dramatically. Larval leg muscles degenerate at the end of larval life, and new adult leg muscles develop during the ensuing pupal stage. Larval leg motoneurons persist, but undergo substantial remodeling of central and peripheral processes. As part of our on‐going investigation of mechanisms underlying the remodeling of motor terminals, we have used antisera generated against Manduca‐specific isoforms of the homophilic adhesion molecule fasciclin II (MFas II) to label motor terminals during metamorphosis. Antisera generated against the glycosyl‐phosphatidylinositol (GPI) ‐linked isoform of MFas II (GPI‐MFas II) labeled the motor nerves at all stages and seemed to be associated with glial cells ensheathing the peripheral nerves. In addition, the anti–GPI‐MFas II antisera labeled regions associated with synaptic boutons at both larval and adult stages. In contrast, antisera generated against a transmembrane isoform of MFas II (TM‐MFas II) only labeled specific neuronal processes at discrete intervals during remodeling. Identified leg motoneurons (such as the femoral depressor motoneuron) expressed detectable levels of TM‐MFas II in their peripheral processes only during phases of motor‐terminal retraction and initial stages of motor‐terminal re‐growth. Putative modulatory neurons (such as the unpaired median neurons), however, expressed TM‐MFas II in their processes during larval stages as well as during remodeling. Use of the isoform‐specific anti‐MFas II antisera provided a novel method for visualizing remodeling of motor terminals during metamorphosis and helped distinguish different components of the motor nerves and neuromuscular junction. J. Comp. Neurol. 434:69–85, 2001.

Reverse Signaling via a Glycosyl-Phosphatidylinositol-Linked Ephrin Prevents Midline Crossing by Migratory Neurons during Embryonic Development in Manduca

We have investigated whether reverse signaling via a glycosyl-phosphatidylinositol (GPI)-linked ephrin controls the behavior of migratory neurons in vivo. During the formation of the enteric nervous system (ENS) in the moth Manduca, ∼300 neurons [enteric plexus (EP) cells] migrate onto the midgut via bilaterally paired muscle bands but avoid adjacent midline regions. As they migrate, the EP cells express a single ephrin ligand (MsEphrin; a GPI-linked ligand), whereas the midline cells express the corresponding Eph receptor (MsEph). Blocking endogenous MsEphrin–MsEph receptor interactions in cultured embryos resulted in aberrant midline crossing by the neurons and their processes. In contrast, activating endogenous MsEphrin on the EP cells with dimeric MsEph-Fc constructs inhibited their migration and outgrowth, supporting a role for MsEphrin-dependent reverse signaling in this system. In short-term cultures, blocking endogenous MsEph receptors allowed filopodia from the growth cones of the neurons to invade the midline, whereas activating neuronal MsEphrin led to filopodial retraction. MsEphrin-dependent signaling may therefore guide the migratory enteric neurons by restricting the orientation of their leading processes. Knocking down MsEphrin expression in the EP cells with morpholino antisense oligonucleotides also induced aberrant midline crossing, consistent with the effects of blocking endogenous MsEphrin–MsEph interactions. Unexpectedly, this treatment enhanced the overall extent of migration, indicating that MsEphrin-dependent signaling may also modulate the general motility of the EP cells. These results demonstrate that MsEphrin–MsEph receptor interactions normally prevent midline crossing by migratory neurons within the developing ENS, an effect that is most likely mediated by reverse signaling through this GPI-linked ephrin ligand.

Eph receptor expression defines midline boundaries for ephrin-positive migratory neurons in the enteric nervous system of Manduca sexta.

Eph receptor tyrosine kinases and their ephrin ligands participate in the control of neuronal growth and migration in a variety of contexts, but the mechanisms by which they guide neuronal motility are still incompletely understood. By using the enteric nervous system (ENS) of the tobacco hornworm Manduca sexta as a model system, we have explored whether Manduca ephrin (MsEphrin; a GPI‐linked ligand) and its Eph receptor (MsEph) might regulate the migration and outgrowth of enteric neurons. During formation of the Manduca ENS, an identified set of ∼300 neurons (EP cells) populates the enteric plexus of the midgut by migrating along a specific set of muscle bands forming on the gut, but the neurons strictly avoid adjacent interband regions. By determining the mRNA and protein expression patterns for MsEphrin and the MsEph receptor and by examining their endogenous binding patterns within the ENS, we have demonstrated that the ligand and its receptor are distributed in a complementary manner: MsEphrin is expressed exclusively by the migratory EP cells, whereas the MsEph receptor is expressed by midline interband cells that are normally inhibitory to migration. Notably, MsEphrin could be detected on the filopodial processes of the EP cells that extended up to but not across the midline cells expressing the MsEph receptor. These results suggest a model whereby MsEphrin‐dependent signaling regulates the response of migrating neurons to a midline inhibitory boundary, defined by the expression of MsEph receptors in the developing ENS. J. Comp. Neurol. 502:175–191, 2007.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aβ) peptide fragments that accumulate in Alzheimers Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2) that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta), we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of our double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, our results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.

Reverse Signaling by Glycosylphosphatidylinositol-Linked Manduca Ephrin Requires a Src Family Kinase to Restrict Neuronal Migration In Vivo

Reverse signaling via glycosylphosphatidylinositol (GPI)-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), whereas adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways.

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration

Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimers disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL–Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin–APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin–APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD. SIGNIFICANCE STATEMENT Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained controversial. This research provides new evidence that members of the Contactin family function as authentic ligands for APP and its orthologs, and that this evolutionarily conserved class of membrane-attached proteins regulates key aspects of APP-dependent migration and outgrowth in the embryonic nervous system. By defining the normal role of Contactin–APP signaling during development, these studies also provide the framework for investigating how the misregulation of Contactin–APP interactions might contribute to neuronal dysfunction in the context of both normal aging and neurodegenerative conditions, including Alzheimers disease.

Role of APP interactions with heterotrimeric G proteins: Physiological functions and pathological consequences

Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth via adenylyl cyclase/PKA-dependent pathways. These reports offer the intriguing perspective that G protein switching might modulate APP-dependent responses in a context-dependent manner. In this review, we provide an up-to-date perspective on the model that APP plays a variety of roles as an atypical G protein-coupled receptor in both the developing and adult nervous system, and we discuss the hypothesis that disruption of these normal functions might contribute to the progressive neuropathologies that typify AD.