Pharmacologic Characterization of Substituted Nitazenes at µ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent µ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human µ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

Enantiomeric Separation, Absolute Configuration by X-ray Crystallographic Analysis, and Functional Evaluation of Enantiomers of the Dual Ligand, SYA0340 at 5-HT1A and 5-HT7A Receptors.

We have previously identified 5-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (SYA0340) as a dual 5-HT1A and 5-HT7 receptor ligand, and we posited such ligands might find utility in the treatment of various CNS related illnesses including cognitive and anxiolytic impairments. However, SYA0340 has a chiral center and its enantiomers may confound the readouts for their functional characteristics. Thus, in this study, we resynthesized SYA0340, separated the enantiomers, identified the absolute configurations, and evaluated their binding affinities and functional characteristics at both the 5-HT1A and 5-HT7A receptors. The results of this study show that the (+)-SYA0340-P1 [specific rotation [α] = +18.4 (deg.mL)/(g.dm)] has a binding affinity constant, Ki = 1.73 ± 0.55 nM at 5-HT1AR and Ki = 2.20 ± 0.33 nM at 5-HT7AR and (-)-SYA0340-P2 [specific rotation [α] = -18.2 (deg.mL)/(g.dm)] has Ki = 1.06 ± 0.32 nM (5-HT1AR) and 4.7 ± 1.1 nM (5-HT7AR). Using X-ray crystallographic techniques, the absolute configuration of the P2 isomer was identified as the S-enantiomer and, therefore, the P1 isomer as the R-enantiomer. Functionally, both SYA0340-P1 (EC50 = 1.12 ± 0.41 nM; Emax = 94.6 ± 3.1%) and SYA0340-P2 (EC50 = 2.21 ± 0.59 nM; Emax = 96.8 ± 5.1%) display similar agonist properties at the 5-HT1AR while both enantiomers display antagonist properties at the 5-HT7AR with P1 (IC50 = 32.1 ± 9.2 nM) displaying over 8 times greater potency as P2 (IC50 = 277 ± 46 nM). Thus, based on the functional evaluation results, SYA0340-P1 is considered as the eutomer of the pair of enantiomers of SYA0340. It is expected that these enantiomers will serve as new pharmacological probes for the 5-HT1A and 5-HT7A receptors.

Fasciclin 2 plays multiple roles in promoting cell migration within the developing nervous system of Manduca sexta.

The coordination of neuronal and glial migration is essential to the formation of most nervous systems, requiring a complex interplay of cell-intrinsic responses and intercellular guidance cues. During the development of the enteric nervous system (ENS) in Manduca sexta (tobacco hornworm), the IgCAM Fasciclin 2 (Fas2) serves several distinct functions to regulate these processes. As the ENS forms, a population of 300 neurons (EP cells) undergoes sequential phases of migration along well-defined muscle pathways on the visceral mesoderm to form a branching Enteric Plexus, closely followed by a trailing wave of proliferating glial cells that enwrap the neurons. Initially, both the neurons and glial cells express a GPI-linked form of Fas2 (GPI-Fas2), which helps maintain cell-cell contact among the pre-migratory neurons and later promotes glial ensheathment. The neurons then switch isoforms, predominantly expressing a combination of transmembrane isoforms lacking an intracellular PEST domain (TM-Fas2 PEST-), while their muscle band pathways on the midgut transiently express transmembrane isoforms containing this domain (TM-Fas2 PEST+). Using intracellular injection protocols to manipulate Fas2 expression in cultured embryos, we found that TM-Fas2 promotes the directed migration and outgrowth of individual neurons in the developing ENS. Concurrently, TM-Fas2 expression by the underlying muscle bands is also required as a substrate cue to support normal migration, while glial expression of GPI-Fas2 helps support their ensheathment of the migratory neurons. These results demonstrate how a specific IgCAM can play multiple roles that help coordinate neuronal and glial migration in the developing nervous system.

Pharmacologic Activity of Substituted Tryptamines at 5-Hydroxytryptamine (5-HT)2A Receptor (5-HT2AR), 5-HT2CR, 5-HT1AR, and Serotonin Transporter.

Novel psychoactive substances, including synthetic substituted tryptamines, represent a potential public health threat. Additionally, some substituted tryptamines are being studied under medical guidance as potential treatments of psychiatric disorders. Characterizing the basic pharmacology of substituted tryptamines will aid in understanding differences in potential for harm or therapeutic use. Using human embryonic kidney cells stably expressing 5-hydroxytryptamine (5-HT)1A, 5-HT2A, and 5-HT2C receptors (5-HT1AR, 5-HT2AR, and 5HT2CR, respectively) or the serotonin transporter (SERT), we measured affinities, potencies and efficacies of 21 substituted tryptamines. With the exception of two 4-acetoxy compounds, substituted tryptamines exhibited affinities and potencies less than one micromolar at the 5-HT2AR, the primary target for psychedelic effects. In comparison, half or more exhibited low affinities/potencies at 5-HT2CR, 5-HT1AR, and SERT. Sorting by the ratio of 5-HT2A to 5-HT2C, 5-HT1A, or SERT affinity revealed chemical determinants of selectivity. We found that although 4-substituted compounds exhibited affinities that ranged across a factor of 100, they largely exhibited high selectivity for 5-HT2ARs versus 5-HT1ARs and 5-HT2CRs. 5-substituted compounds exhibited high affinities for 5-HT1ARs, low affinities for 5-HT2CRs, and a range of affinities for 5-HT2ARs, resulting in selectivity for 5-HT2ARs versus 5-HT2CRs but not versus 5-HT1ARs. Additionally, a number of psychedelics bound to SERT, with non-ring-substituted tryptamines most consistently exhibiting binding. Interestingly, substituted tryptamines and known psychedelic standards exhibited a broad range of efficacies, which were lower as a class at 5-HT2ARs compared with 5-HT2CRs and 5-HT1ARs. Conversely, coupling efficiency/amplification ratio was highest at 5-HT2ARs in comparison with 5-HT2CRs and 5-HT1ARs. SIGNIFICANCE STATEMENT: Synthetic substituted tryptamines represent both potential public health threats and potential treatments of psychiatric disorders. The substituted tryptamines tested differed in affinities, potencies, and efficacies at 5-hydroxytryptamine (5-HT)2A, 5-HT2C, and 5HT1A receptors and the serotonin transporter (SERT). Several compounds were highly selective for and coupled very efficiently downstream of 5-HT2A versus 5-HT1A and 5-HT2C receptors, and some bound SERT. This basic pharmacology of substituted tryptamines helps us understand the pharmacologic basis of their potential for harm and as therapeutic agents.

A highly D3R-selective and efficacious partial agonist (S)-ABS01-113 compared to its D3R-selective antagonist enantiomer (R)-ABS01-113 as potential treatments for opioid use disorder.

The non-medical use of opioids has become a national crisis in the USA. Developing non-opioid pharmacotherapies for controlling this opioid epidemic is urgent. Dopamine D3 receptor (D3R) antagonists and low efficacy partial agonists have shown promising profiles in animal models of opioid use disorders (OUD). However, to date, advancement to human studies has been limited. Here we report the effects of (S)- and (R)-enantiomers of (±)-ABS01-113, structural analogs of the D3R partial agonist, (±)-VK4-40, in which the 3-OH in the linking chain is replaced by 3-F group. (S)- and (R)-ABS01-113 are identical in chemical structure but with opposite chirality. In vitro receptor binding and functional assays indicate that (S)-ABS01-113 is an efficacious (55%) and potent (EC50 = 7.6 ± 3.9 nM) D3R partial agonist, while the (R)-enantiomer is a potent D3R antagonist (IC50 = 11.4 nM). Both (S)- and (R)-ABS01-113 bind with high affinity to D3R (Ki = 0.84 ± 0.16 and 0.37 ± 0.06 nM, respectively); however, the (S)-enantiomer is more D3/D2-selective (>1000-fold). Pharmacokinetic analyses indicate that both enantiomers display excellent oral bioavailability and high brain penetration. Systemic administration of (S)- or (R)-ABS01-113 alone failed to alter open-field locomotion in male rats and mice. Interestingly, pretreatment with (S)- or (R)-ABS01-113 attenuated heroin-enhanced hyperactivity, heroin self-administration, and (heroin + cue)-induced reinstatement of drug-seeking behavior. Together, these findings reveal that both enantiomers, particularly the highly selective and efficacious D3R partial agonist (S)-ABS01-113, demonstrate promising translational potential for the treatment of OUD.

New dual 5-HT1A and 5-HT7 receptor ligands derived from SYA16263.

We have previously reported that dual 5-HT1A and 5-HT7 receptor ligands might find utility as treatment options for various CNS related conditions including cognitive and anxiolytic impairments. We have also more recently reported that SYA16263 has antipsychotic-like properties with an absence of catalepsy in animal models ascribed to its ability to recruit ß-arrestin to the D2 receptor. However, SYA16263 also binds with very high affinity to 5-HT1AR (Ki = 1.1 nM) and a moderate affinity at 5-HT7R (Ki = 90 nM). Thus, it was of interest to exploit its pharmacophore elements in designing new dual receptor ligands. Using SYA16263 as the lead molecule, we have conducted a limited structure-affinity relationship (SAFIR) study by modifying various structural elements in the arylalkyl moiety, resulting in the identification of a new dual 5-HT1AR and 5-HT7R ligand, 6-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (21), which unlike SYA16263, has a sub-nanomolar (5-HT1AR, Ki = 0.74 nM) and a low nanomolar (5-HT7R, Ki = 8.4 nM) affinity for these receptors. Interestingly, 21 is a full agonist at 5-HT1AR and antagonist at the 5-HT7R, functional characteristics which point to its potential as an antidepressant agent.

Fentanyl but not Morphine Interacts with Nonopioid Recombinant Human Neurotransmitter Receptors and Transporters.

Synthetic opioids, including fentanyl and its analogs, have therapeutic efficacy in analgesia and anesthesia. However, their illicit use in the United States has increased and contributed to the number one cause of death for adults 18-50 years old. Fentanyl and the heroin metabolite morphine induce respiratory depression that can be treated with the µ opioid receptor (MOR) antagonist naloxone. With higher or more rapid dosing, fentanyl, more than morphine, causes chest wall rigidity and can also induce rapid onset laryngospasm. Because non-MORs could mediate differing clinical manifestations, we examined the interactions of fentanyl and morphine at recombinant human neurotransmitter transporters, G protein-coupled receptors, and the N-methyl-D-aspartate glutamate receptor. Both drugs were agonists at MOR, κ, and δ opioid receptors. Morphine had little or no affinity at other human receptors and transporters (K i or IC50 value >100 µM). However, fentanyl had K i values of 1407 and 1100 nM at α 1A and α 1B adrenoceptor subtypes, respectively, and K i values of 1049 and 1670 nM at dopamine D4.4 and D1 receptor subtypes, respectively; it also blocked [3H]neurotransmitter uptake by the vesicular monoamine transporter 2 (IC50 = 911 nM). Pharmacokinetic models indicate that these Ki and IC50 values are pharmacologically relevant. Fentanyl had little affinity for other receptors or transporters. Thus, noradrenergic disposition at specific receptor subtypes in relevant organs may play a role in respiratory and cardiothoracic effects of fentanyl. Data suggest that less selective fentanyl receptor pharmacology could play a role in the different clinical effects of morphine compared with fentanyl, including fentanyl-induced deaths after illicit use. SIGNIFICANCE STATEMENT: The synthetic opioid fentanyl induces different clinical effects, including rapid onset muscular rigidity, vocal cord closure, and rapid death, than the heroin metabolite morphine. Our data indicate for the first time that the two drugs have very different effects at recombinant human neurotransmitter receptors and transporters that might explain those clinical differences.

Activation of Trace Amine-Associated Receptor 1 Stimulates an Antiapoptotic Signal Cascade via Extracellular Signal-Regulated Kinase 1/2.

Methamphetamine (MA) is highly addictive and neurotoxic, causing cell death in humans and in rodent models. MA, along with many of its analogs, is an agonist at the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 activation protects against MA-induced degeneration of dopaminergic neurons, suggesting that TAAR1 plays a role in regulating MA-induced neurotoxicity. However, the mechanisms involved in TAAR1's role in neurotoxicity and cell death have not been described in detail. In this study, we investigated the apoptosis pathway in Taar1 wild-type (WT) and knockout (KO) mice and in cells expressing the recombinant receptor. Bcl-2, an antiapoptotic protein, was upregulated ∼3-fold in the midbrain area (substantial nigra and ventral tegmental area) in Taar1 KO compared with WT mice, and MA significantly increased Bcl-2 expression in WT mice but decreased Bcl-2 expression in KO mice. The proapoptotic protein Bax did not differ across genotype or in response to MA. Bcl-2 expression was significantly upregulated by the TAAR1 agonist RO5166017 ((S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine) in cells expressing the recombinant mouse TAAR1. Additionally, activation of TAAR1 by RO5166017 increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and protein kinase B (AKT), but only inhibition of ERK1/2 phosphorylation prevented TAAR1-induced increases in Bcl-2 levels, indicating that TAAR1 activation increases Bcl-2 through an ERK1/2-dependent pathway. All changes to ERK1/2 pathway intermediates were blocked by the TAAR1 antagonist, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl) benzamide. These findings suggest that TAAR1 activation protects against MA-induced cell apoptosis and TAAR1 may play a role in cell death in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Methamphetamine stimulates TAAR1, a G protein-coupled receptor. The role and mechanisms for TAAR1 in methamphetamine-induced neurotoxicity are not known. Here, we report that, in genetic mouse models and cells expressing the recombinant receptor, TAAR1 activates the ERK1/2 pathway but not the AKT pathway to upregulate the antiapoptotic protein Bcl-2, which protects cells from drug-induced toxicity.

Structure-activity relationships of bath salt components: substituted cathinones and benzofurans at biogenic amine transporters.

RATIONALE: New psychoactive substances (NPSs), including substituted cathinones and other stimulants, are synthesized, sold on the Internet, and ingested without knowledge of their pharmacological activity and/or toxicity. In vitro pharmacology plays a role in therapeutic drug development, drug-protein in silico interaction modeling, and drug scheduling. OBJECTIVES: The goal of this research was to determine mechanisms of action that may indicate NPS abuse liability. METHODS: Affinities to displace the radioligand [125I]RTI-55 and potencies to inhibit [3H]neurotransmitter uptake for 22 cathinones, 6 benzofurans and another stimulant were characterized using human embryonic kidney cells stably expressing recombinant human transporters for dopamine, norepinephrine, or serotonin (hDAT, hNET, or hSERT, respectively). Selected compounds were tested for potencies and efficacies at inducing [3H]neurotransmitter release via the transporters. Computational modeling was conducted to explain plausible molecular interactions established by NPS and transporters. RESULTS: Most α-pyrrolidinophenones had high hDAT potencies and selectivities in uptake assays, with hDAT/hSERT uptake selectivity ratios of 83-360. Other substituted cathinones varied in their potencies and selectivities, with N-ethyl-hexedrone and N-ethyl-pentylone having highest hDAT potencies and N-propyl-pentedrone having highest hDAT selectivity. 4-Cl-ethcathinone and 3,4-methylenedioxy-N-propylcathinone had higher hSERT selectivity. Benzofurans generally had low hDAT selectivity, especially 1-(2,3-dihydrobenzofuran-5-yl)-N-methylpropan-2-amine, with 25-fold higher hSERT potency. Consistent with this selectivity, the benzofurans were releasers at hSERT. Modeling indicated key amino acids in the transporters' binding pockets that influence drug affinities. CONCLUSIONS: The α-pyrrolidinophenones, with high hDAT selectivity, have high abuse potential. Lower hDAT selectivity among benzofurans suggests similarity to methylenedioxymethamphetamine, entactogens with lower stimulant activity.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development.

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aß) peptide fragments that accumulate in Alzheimer's 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 Aß-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.

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration.

UNLABELLED: Amyloid precursor protein (APP) was originally identified as the source of ß-amyloid peptides that accumulate in Alzheimer's 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 Alzheimer's disease.

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 ß-amyloid fragments (Aß) that accumulate in Alzheimer's 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 Aß. 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.

Amyloid precursor proteins are protective in Drosophila models of progressive neurodegeneration.

The processing of Amyloid Precursor Proteins (APPs) results in several fragments, including soluble N-terminal ectodomains (sAPPs) and C-terminal intracellular domains (AICD). sAPPs have been ascribed neurotrophic or neuroprotective functions in cell culture, although ß-cleaved sAPPs can have deleterious effects and trigger neuronal cell death. Here we describe a neuroproprotective function of APP and fly APPL (Amyloid Precursor Protein-like) in vivo in several Drosophila mutants with progressive neurodegeneration. We show that expression of the N-terminal ectodomain is sufficient to suppress the progressive degeneration in these mutants and that the secretion of the ectodomain is required for this function. In addition, a protective effect is achieved by expressing kuzbanian (which has α-secretase activity) whereas expression of fly and human BACE aggravates the phenotypes, suggesting that the protective function is specifically mediated by the α-cleaved ectodomain. Furthermore, genetic and molecular studies suggest that the N-terminal fragments interact with full-length APPL activating a downstream signaling pathway via the AICD. Because we show protective effects in mutants that affect different genes (AMP-activated protein kinase, MAP1b, rasGAP), we propose that the protective effect is not due to a genetic interaction between APPL and these genes but a more general aspect of APP proteins. The result that APP proteins and specifically their soluble α-cleaved ectodomains can protect against progressive neurodegeneration in vivo provides support for the hypothesis that a disruption of the physiological function of APP could play a role in the pathogenesis of Alzheimer's Disease.

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

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 (Ca(2+)) 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 Ca(2+) 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 ß-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca(2+) 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 Ca(2+) regulatory machinery and that therefore might be beneficial in the treatment of AD.

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.

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 approximately 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.